Double stranded constructs comprising one or more short strands hybridized to a longer strand

ABSTRACT

The present invention provides compositions comprising an antisense oligomeric compound hybridized to at least one shorter sense oligomeric compound. The sense oligomeric compounds can be covalently linked to the antisense oligomeric compounds. At least a portion of the antisense oligomeric compound is complementary to and hybridizes with a nucleic acid target. Methods for modulating gene expression are also provided using the compositions of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit to U.S. Provisional ApplicationSer. No. 60/551,670 filed Mar. 8, 2004, which is incorporated herein inits entirety.

FIELD OF THE INVENTION

The present invention provides compositions comprising an antiseneseoligomeric comound and at least one shorter sense oligomeric compoundwherein each sense oligomeric compound hybridizes with the antisenseoligomeric comound and wherein the antisense oligomeric compound hascomplementarity to and hybridizes to a target nucleic acid and methodsfor their use in modulating gene expression. In preferred embodimentscompositions of the present invention hybridize to a portion of a targetRNA resulting in loss of normal function of the target RNA.

BACKGROUND OF THE INVENTION

In many species, introduction of double-stranded RNA (dsRNA) inducespotent and specific gene silencing. This phenomenon occurs in bothplants and animals and has roles in viral defense and transposonsilencing mechanisms. This phenomenon was originally described more thana decade ago by researchers working with the petunia flower. Whiletrying to deepen the purple color of these flowers, Jorgensen et al.introduced a pigment-producing gene under the control of a powerfulpromoter. Instead of the expected deep purple color, many of the flowersappeared variegated or even white. Jorgensen named the observedphenomenon “cosuppression”, since the expression of both the introducedgene and the homologous endogenous gene was suppressed (Napoli et al.,Plant Cell, 1990, 2, 279-289; Jorgensen et al., Plant Mol. Biol., 1996,31, 957-973).

Cosuppression has since been found to occur in many species of plants,fungi, and has been particularly well characterized in Neurosporacrassa, where it is known as “quelling” (Cogoni and Macino, Genes Dev.2000, 10, 638-643; Guru, Nature, 2000, 404, 804-808).

The first evidence that dsRNA could lead to gene silencing in animalscame from work in the nematode, Caenorhabditis elegans. In 1995,researchers Guo and Kemphues were attempting to use antisense RNA toshut down expression of the par-1 gene in order to assess its function.As expected, injection of the antisense RNA disrupted expression ofpar-1, but quizzically, injection of the sense-strand control alsodisrupted expression (Guo and Kempheus, Cell, 1995, 81, 611-620). Thisresult was a puzzle until Fire et al. injected dsRNA (a mixture of bothsense and antisense strands) into C. elegans. This injection resulted inmuch more efficient silencing than injection of either the sense or theantisense strands alone. Injection of just a few molecules of dsRNA percell was sufficient to completely silence the homologous gene'sexpression. Furthermore, injection of dsRNA into the gut of the wormcaused gene silencing not only throughout the worm, but also in firstgeneration offspring (Fire et al., Nature, 1998, 391, 806-811).

The potency of this phenomenon led Timmons and Fire to explore thelimits of the dsRNA effects by feeding nematodes bacteria that had beenengineered to express dsRNA homologous to the C. elegans unc-22 gene.Surprisingly, these worms developed an unc-22 null-like phenotype(Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001,263, 103-112). Further work showed that soaking worms in dsRNA was alsoable to induce silencing (Tabara et al., Science, 1998, 282, 430-431).PCT publication WO 01/48183 discloses methods of inhibiting expressionof a target gene in a nematode worm involving feeding to the worm a foodorganism which is capable of producing a double-stranded RNA structurehaving a nucleotide sequence substantially identical to a portion of thetarget gene following ingestion of the food organism by the nematode, orby introducing a DNA capable of producing the double-stranded RNAstructure (Bogaert et al., 2001).

The posttranscriptional gene silencing defined in Caenorhabditis elegansresulting from exposure to double-stranded RNA (dsRNA) has since beendesignated as RNA interference (RNAi). This term has come to generalizeall forms of gene silencing involving dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels; unlikeco-suppression, in which transgenic DNA leads to silencing of both thetransgene and the endogenous gene. Introduction of exogenousdouble-stranded RNA (dsRNA) into Caenorhabditis elegans has been shownto specifically and potently disrupt the activity of genes containinghomologous sequences. Montgomery et al. suggests that the primaryinterference effects of dsRNA are post-transcriptional; this conclusionbeing derived from examination of the primary DNA sequence afterdsRNA-mediated interference a finding of no evidence of alterationsfollowed by studies involving alteration of an upstream operon having noeffect on the activity of its downstream gene. These results argueagainst an effect on initiation or elongation of transcription. Finallythey observed by in situ hybridization, that dsRNA-mediated interferenceproduced a substantial, although not complete, reduction in accumulationof nascent transcripts in the nucleus, while cytoplasmic accumulation oftranscripts was virtually eliminated. These results indicate that theendogenous mRNA is the primary target for interference and suggest amechanism that degrades the targeted mRNA before translation can occur.It was also found that this mechanism is not dependent on the SMGsystem, an mRNA surveillance system in C. elegans responsible fortargeting and destroying aberrant messages. The authors further suggesta model of how dsRNA might function as a catalytic mechanism to targethomologous mRNAs for degradation. (Montgomery et al., Proc. Natl. Acad.Sci. USA, 1998, 95, 15502-15507).

Recently, the development of a cell-free system from syncytialblastoderm Drosophila embryos that recapitulates many of the features ofRNAi has been reported. The interference observed in this reaction issequence specific, is promoted by dsRNA but not single-stranded RNA,functions by specific mRNA degradation, and requires a minimum length ofdsRNA. Furthermore, preincubation of dsRNA potentiates its activitydemonstrating that RNAi can be mediated by sequence-specific processesin soluble reactions (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).

In subsequent experiments, Tuschl et al, using the Drosophila in vitrosystem, demonstrated that 21- and 22-nt RNA fragments are thesequence-specific mediators of RNAi. These fragments, which they termedshort interfering RNAs (siRNAs) were shown to be generated by an RNaseIII-like processing reaction from long dsRNA. They also showed thatchemically synthesized siRNA duplexes with overhanging 3′ ends mediateefficient target RNA cleavage in the Drosophila lysate, and that thecleavage site is located near the center of the region spanned by theguiding siRNA. In addition, they suggest that the direction of dsRNAprocessing determines whether sense or antisense target RNA can becleaved by the siRNA-protein complex (Elbashir et al., Genes Dev., 2001,15, 188-200). Further characterization of the suppression of expressionof endogenous and heterologous genes caused by the 21-23 nucleotidesiRNAs have been investigated in several mammalian cell lines, includinghuman embryonic kidney (293) and HeLa cells (Elbashir et al., Nature,2001, 411, 494-498).

Most recently, Tijsterman et al. have shown that, in fact,single-stranded RNA oligomers of antisense polarity can be potentinducers of gene silencing. As is the case for co-suppression, theyshowed that antisense RNAs act independently of the RNAi genes rde-1 andrde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD boxRNA helicase, mut-14. According to the authors, their data favor thehypothesis that gene silencing is accomplished by RNA primer extensionusing the mRNA as template, leading to dsRNA that is subsequentlydegraded suggesting that single-stranded RNA oligomers are ultimatelyresponsible for the RNAi phenomenon (Tijsterman et al., Science, 2002,295, 694-697).

Several recent publications have described the structural requirementsfor the dsRNA trigger required for RNAi activity. Recent reports haveindicated that ideal dsRNA sequences are 21 nt in length containing 2 nt3′-end overhangs (Elbashir et al, EMBO (2001), 20, 6877-6887, SabineBrantl, Biochimica et Biophysica Acta, 2002, 1575, 15-25.) In thissystem, substitution of the 4 nucleosides from the 3′-end with2′-deoxynucleosides has been demonstrated to not affect activity. On theother hand, substitution with 2′deoxynucleosides or 2′-OMe-nucleosidesthroughout the sequence (sense or antisense) was shown to be deleteriousto RNAi activity.

Investigation of the structural requirements for RNA silencing in C.elegans has demonstrated modification of the internucleotide linkage(phosphorothioate) to not interfere with activity (Parrish et al.,Molecular Cell, 2000, 6, 1077-1087.) It was also shown by Parrish etal., that chemical modification like 2′-amino or 5′-iodouridine are welltolerated in the sense strand but not the antisense strand of the dsRNAsuggesting differing roles for the 2 strands in RNAi. Base modificationsuch as guanine to inosine (where one hydrogen bond is lost) has beendemonstrated to decrease RNAi activity independently of the position ofthe modification (sense or antisense). Some “position independent” lossof activity has been observed following the introduction of mismatchesin the dsRNA trigger. Some types of modifications, for exampleintroduction of sterically demanding bases such as 5-iodoU, have beenshown to be deleterious to RNAi activity when positioned in theantisense strand, whereas modifications positioned in the sense strandwere shown to be less detrimental to RNAi activity. As was the case forthe 21 nt dsRNA sequences, RNA-DNA heteroduplexes did not serve astriggers for RNAi. However, dsRNA containing 2′-F-2′-deoxynucleosidesappeared to be efficient in triggering RNAi response independent of theposition (sense or antisense) of the 2′-F-2′-deoxynucleosides.

In one experiment the reduction of gene expression was studied usingelectroporated dsRNA and a 25 mer morpholino in post-implantation mouseembryos (Mellitzer et al., Mehanisms of Development, 2002, 118, 57-63).The morpholino oligomer did show activity but was not as effective asthe dsRNA.

A number of PCT applications have recently been published that relate tothe RNAi phenomenon. These include: PCT publication WO 00/44895; PCTpublication WO 00/49035; PCT publication WO 00/63364; PCT publication WO01/36641; PCT publication WO 01/36646; PCT publication WO 99/32619; PCTpublication WO 00/44914; PCT publication WO 01/29058; and PCTpublication WO 01/75164.

U.S. Pat. Nos. 5,898,031 and 6,107,094, each of which is commonly ownedwith this application and each of which is herein incorporated byreference, describe certain oligonucleotide having RNA like properties.When hybridized with RNA, these olibonucleotides serve as substrates fora dsRNase enzyme with resultant cleavage of the RNA by the enzyme.

In another recently published paper (Martinez et al., Cell, 2002, 110,563-574) it was shown that double stranded as well as single strandedsiRNA resides in the RNA-induced silencing complex (RISC) together witheIF2C1 and eIf2C2 (human GERp950 Argonaute proteins. The activity of5′-phosphorylated single stranded siRNA was comparable to the doublestranded siRNA in the system studied. In a related study, the inclusionof a 5′-phosphate moiety was shown to enhance activity of siRNA's invivo in Drosophilia embryos (Boutla, et al., Curr. Biol., 2001, 11,1776-1780). In another study, it was reported that the 5′-phosphate wasrequired for siRNA function in human HeLa cells (Schwarz et al.,Molecular Cell, 2002, 10, 537-548).

In one recently published paper the authors claim that inclusion of2′-O-methyl groups into the sense, antisense or both the sense andantisense strands of a siRNA showed greatly reduced activity (Chiu,Ya-Lin and Rana, Tariq, M., RNA, 2003, 9, 1034-1048).

Recently, it has become clear that non-coding RNA genes producefunctional RNA molecules with important roles in regulation of geneexpression, as well as in developmental timing, viral surveillance, andimmunity. Not only the classic transfer RNAs (tRNAs) and ribosomal RNAs(rRNAs), but also small nuclear RNAs (snRNAs), small nucleolar RNAs(snoRNAs), small interfering RNAs (siRNAs), tiny non-coding RNAs(tncRNAs) and microRNAs (miRNAs) are now known to act in diversecellular processes such as chromosome maintenance, gene imprinting,pre-mRNA splicing, guiding RNA modifications, transcriptionalregulation, and the control of mRNA translation (Eddy, Nature ReviewsGenetics, 2001, 2, 919-929; Kawasaki and Taira, Nature, 2003, 423,838-842). RNA-mediated processes are now also believed to directheterochromatin formation, genome rearrangements, and DNA elimination(Cerutti, Trends in Genetics, 2003, 19, 39-46; Couzin, Science, 2002,298, 2296-2297)

A large class of small non-coding RNAs known as microRNAs (miRNAs) playsa role in regulation of gene expression. In C. elegans, Drosophila, andhumans, miRNAs are predicted to function as endogenouspost-transcriptional gene regulators. The founding members of the miRNAfamily are transcribed by the C. elegans genes let-7 and lin-4, and werefirst dubbed “short temporal RNAs” (stRNAs). The let-7 and lin-4 miRNAsact as antisense translational repressors of messenger RNAs that encodeproteins crucial to the heterochronic developmental timing pathway innematode larva. (Ambros, Cell, 2001, 107, 823-826; Ambros et al.,Current Biology, 2003, 13, 807-818).

Mature miRNAs originate from long endogenous primary transcripts(pri-miRNAs) that are often hundreds of nucleotides in length (Lee, etal., EMBO J., 2002, 21(17), 4663-4670). These pri-miRNAs are processedby a nuclear enzyme in the RNase III family known as Drosha, intoapproximately 70 nucleotide-long pre-miRNAs (also known as stem-loop,hairpin or foldback precursors) which are subsequently processed by theDicer RNase into mature miRNAs (Lee, et al., Nature, 2003, 425,415-419). The current model is that the pri-miRNA transcript isprocessed by Drosha in the nucleus, and the pre-miRNA hairpin precursoris exported from the nucleus and cleaved by Dicer in the cytosol toyield a double-stranded intermediate (similar in size to siRNAs), butonly one strand of this short-lived intermediate accumulates as themature miRNA (Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel,Plant Physiology, 2003, 132, 709-717; Shi, Trends in Genetics, 2003, 19,9-12).

miRNAs, like siRNAs, are processed by the Dicer enzyme, areapproximately the same length, and possess the characteristic5′-phosphate and 3′-hydroxyl termini. The miRNAs are also incorporatedinto a ribonucleoprotein complex, the miRNP, which is similar to theRNA-induced silencing complex, i.e., the RISC complex, through whichsiRNAs act (Bartel and Bartel, Plant Physiology, 2003, 132, 709-717).More than 200 different miRNAs have been identified in plants andanimals (Ambros et al., Current Biology, 2003, 13, 807-818).

While siRNAs cause gene silencing by target RNA cleavage anddegradation, miRNAs are believed to direct translational repression,primarily. This functional difference may be related to the fact thatmiRNAs tolerate multiple base pair mismatches whereas siRNAs areperfectly complementary to their target substrates (Ambros et al.,Current Biology, 2003, 13, 807-818; Bartel and Bartel, Plant Physiology,2003, 132, 709-717; Shi, Trends in Genetics, 2003, 19, 9-12).

Like the RNAse H pathway, the RNA interference pathway of antisensemodulation of gene expression is an effective means for modulating thelevels of specific gene products and may therefore prove to be uniquelyuseful in a number of therapeutic, diagnostic, and research applicationsinvolving gene silencing. The present invention therefore furtherprovides compositions useful for modulating gene expression pathways,including those relying on an antisense mechanism of action such as RNAinterference and dsRNA enzymes as well as non-antisense mechanisms. Onehaving skill in the art, once armed with this disclosure will be able,without undue experimentation, to identify preferred compositions forthese uses.

SUMMARY OF THE INVENTION

The present invention provides novel compositions each comprising anantisense oligomeric compound and at least one shorter sense oligomericcompound that is at least partially hybridized to and forms a duplexregion with the antisense oligomeric compound. In one embodiment thecomposition of comprises at least two sense oligomeric compounds thatare each at least partially complementary to the antisense oligomericcompound forming duplex regions between the at least two sense strandsand the antisense strand. In a further embodiment the sense oligomericcompound(s) are substantially or completely hybridized to the antisenseoligomeric compound. In another embodiment the double stranded portionof the complex is continuous and comprises two sense and one antisenseoligomeric compounds. In another embodiment two sense strands form twoseparate duplexed regions having an unhybridized single stranded regionof the antisense oligomeric compound between them.

In one embodiment the composition further comprises at least onecovalent linkage between the antisense oligomeric compound and the atleast one sense oligomeric compound. In a further embodiment one senseoligomeric compound is covalently linked to the 5′-termini of theantisense oligomeric compound. In another embodiment one senseoligomeric compound is covalently linked to the 3′-termini of theantisense oligomeric compound. In an even further embodiment one senseoligomeric compound is covalently linked to the 5′-termini of theantisense oligomeric compound and another sense oligomeric compound iscovalently linked to the 3′-termini of the antisense oligomericcompound.

In one embodiment the each of the covalent linkages independentlycomprises a sequence of non-hybridizing linked nucleosides or abifunctional linking group. In a preferred embodiment the bifunctionallinking group comprises a polyethylene glycol linking group.

In one embodiment the antisense oligomeric compounds and senseoligomeric compounds comprise pluralities of linked nucleosides linkedby internucleoside linking groups. In a preferred embodiment thecomformational geometry of each of the linked nucleosides is 3′-endowherein the 3′-endo comformational geometry can derive from multipledissimilar nucleoside modifications such as 2′-modified, ring modifiedor bridging (e.g. bicyclic sugars). In another embodiment each of thenucleosides having 3′-endo conformational geometry comprises a2′-substitutuent group. In one embodiment the each 2′-substituent groupis, independently, —F, —OH, —O—CH₂CH₂—O—CH₃, —O—CH₃, —O—CH₂—CH═CH₂ or—O—CH₂—CH—CH₂—NH(R_(j)) where R_(j) is H or C₁-C₁₀ alkyl with apreferred list of 2′-substituent groups including —F, —OH,—O—CH₂CH₂—O—CH₃ or —O—CH₃.

In one embodiment the 3′-endo conformation geometry is due to thenucleosides having a 2′-O—(CH₂)_(n)-4′ bridge (n=1 or 2). In anotherembodiment the 3′-endo conformation geometry is due to the nucleosideshaving a 4′-S ring modification. In another embodiment the compositionsinclude at least one native RNA nucleoside (β-D-ribonucleoside). Inanother embodiment the nucleosides of the compositions are each modifiednucleosides having 3′-endo modified nucleosides that are other than2′-OH groups (2′-H also excluded as not a 3′-endo configuration).

In one embodiment each of the internucleoside linking groups isindependently a phosphodiester or a phosphorothioate internucleosidelinking group.

In one embodiment the antisense and sense oligomeric compoundsoptionally comprises a terminal phosphate group, a terminal stabilizingor capping group, a 3′-overhang or a conjugate group. In a preferredembodiment the 5′-terminus of the antisense oligomeric compoundcomprises a 5′-phosphate group.

In a preferred embodiment the antisense oligomeric compounds are fromabout 15 to about 30 nucleobases in length and each of the senseoligomeric compound are from about 2 to about 12 nucleobases in length.

In one embodiment of the present invention the at least one senseoligomeric compound is covalently linked to the antisense oligomericcompound. A preferred group of covalent linkages includes a sequence ofnon-hybridizing linked nucleosides or a bifunctional linking group withpreferred bifunctional linking groups including those comprising an(ethylene glycol)₁₋₆ linking group. A further bifunctional linking groupincludes —O—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂— a (ethylene glycol)₃ linkinggroup.

In one embodiment of the present invention the composition comprises atleast two sense oligomeric compounds wherein the sense compounds eachhave a region that is complementary to and forms a duplex region withthe antisense oligomeric compound. In a further embodiment the at leasttwo sense oligomeric compounds are covalently linked to the antisenseoligomeric compound.

In one embodiment of the present invention each of the antisenseoligomeric compounds and each of the sense oligomeric compoundscomprises a plurality of linked nucleosides linked by internucleosidelinking groups.

In one embodiment of the present invention each of the nucleosides ofthe antisense oligomeric compound is a nucleoside having 3′-endoconformational geometry. In a preferred embodiment the nucleosideshaving 3′-endo conformational geometry each comprise a 2′-substitutuentgroup. One group of preferred 2′-substituent groups includes —F, —OH,—O—CH₂CH₂—O—CH₃, —O—CH₃, —O—CH₂—CH═CH₂ and groups having one of formulaI_(a) or II_(a):

wherein:

-   -   R_(b) is O, S or NH;    -   R_(d) is a single bond, O, S or C(═O);    -   R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),        N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);    -   R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;    -   R_(r) is —R_(x)—R_(y);    -   each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,        C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl,        substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or        unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a        chemical functional group or a conjugate group, wherein the        substituent groups are selected from hydroxyl, amino, alkoxy,        carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,        alkyl, aryl, alkenyl and alkynyl;    -   or optionally, R_(u) and R_(v), together form a phthalimido        moiety with the nitrogen atom to which they are attached;    -   each R_(w) is, independently, substituted or unsubstituted        C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);    -   R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);    -   R_(x) is a bond or a linking moiety;    -   R_(y) is a chemical functional group, a conjugate group or a        solid support medium;    -   each R_(m) and R_(n) is, independently, H, a nitrogen protecting        group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, wherein the substituent groups are selected from        hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺,        N(R_(u))(R_(v)), guanidino and acyl where the acyl is an acid        amide or an ester;    -   or R_(m) and R_(n), together, are a nitrogen protecting group,        are joined in a ring structure that optionally includes an        additional heteroatom selected from N and O or are a chemical        functional group;    -   R_(i) is OR_(z), SR_(z), or N(R_(z))₂;    -   each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);    -   R_(f), R_(g) and R_(h) comprise a ring system having from about        4 to about 7 carbon atoms or having from about 3 to about 6        carbon atoms and 1 or 2 heteroatoms wherein the heteroatoms are        selected from oxygen, nitrogen and sulfur and wherein the ring        system is aliphatic, unsaturated aliphatic, aromatic, or        saturated or unsaturated heterocyclic;    -   R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,        alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to        about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,        N(R_(k))(R_(m)) OR_(k), halo, SR_(k) or CN;    -   m_(a) is 1 to about 10;    -   each mb is, independently, 0 or 1;    -   mc is 0 or an integer from 1 to 10;    -   md is an integer from 1 to 10;    -   me is from 0, 1 or 2; and    -   provided that when mc is 0, md is greater than 1.

A further group of 2′-substituent groups includes —F, —OH,—O—CH₂CH₂—O—CH₃, —O—CH₃, —O—CH₂—CH═CH₂ or —O—CH₂—CH—CH₂—NH(R_(j)) whereR_(j) is H and C₁-C₁₀ alkyl. With a further list of 2′-substituentgroups including —F, —OH, —O—CH₂CH₂—O—CH₃ or —O—CH₃.

In one embodiment of the present invention each of the nucleosides ofthe sense oligomeric compound is a nucleoside having 3′-endoconformational geometry. In another embodiment each of the nucleosidehaving 3′-endo conformational geometry comprises a 2′-substitutuentgroup. One preferred 2′-substitutuent groups is 2′-OH. Another group ofpreferred 2′-substituent groups includes —F, —OH, —O—CH₂CH₂—O—CH₃,—O—CH₃, —O—CH₂—CH═CH₂ and a group having one of formula I_(a) or II_(a):

wherein:

-   -   R_(b) is O, S or NH;    -   R_(d) is a single bond, O, S or C(═O);    -   R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),        N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);    -   R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;    -   R_(r) is —R_(x)—R_(y);    -   each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,        C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl,        substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or        unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a        chemical functional group or a conjugate group, wherein the        substituent groups are selected from hydroxyl, amino, alkoxy,        carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,        alkyl, aryl, alkenyl and alkynyl;    -   or optionally, R_(u) and R_(v), together form a phthalimido        moiety with the nitrogen atom to which they are attached;    -   each R_(w) is, independently, substituted or unsubstituted        C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);    -   R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);    -   R_(x) is a bond or a linking moiety;    -   R_(y) is a chemical functional group, a conjugate group or a        solid support medium;    -   each R_(m) and R_(n) is, independently, H, a nitrogen protecting        group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, wherein the substituent groups are selected from        hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺,        N(R_(u))(R_(v)), guanidino and acyl where the acyl is an acid        amide or an ester;    -   or R_(m) and R_(n), together, are a nitrogen protecting group,        are joined in a ring structure that optionally includes an        additional heteroatom selected from N and O or are a chemical        functional group;    -   R_(i) is OR_(z), SR_(z), or N(R_(z))₂;    -   each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);    -   R_(f), R_(g) and R_(h) comprise a ring system having from about        4 to about 7 carbon atoms or having from about 3 to about 6        carbon atoms and 1 or 2 heteroatoms wherein the heteroatoms are        selected from oxygen, nitrogen and sulfur and wherein the ring        system is aliphatic, unsaturated aliphatic, aromatic, or        saturated or unsaturated heterocyclic;    -   N_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,        alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to        about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,        N(R_(k))(R_(m)) OR_(k), halo, SR_(k) or CN;    -   m_(a) is 1 to about 10;    -   each mb is, independently, 0 or 1;    -   mc is 0 or an integer from 1 to 10;    -   md is an integer from 1 to 10;    -   me is from 0, 1 or 2; and    -   provided that when mc is 0, md is greater than 1.

A further group of 2′-substituent groups includes —F, —OH,—O—CH₂CH₂—O—CH₃, —O—CH₃, —O—CH₂—CH═CH₂ or —O—CH₂—CH—CH₂—NH(R_(j)) whereR_(j) is H and C₁-C₁₀ alkyl. With a further list of 2′-substituentgroups including —F, —OH, —O—CH₂CH₂—O—CH₃ or —O—CH₃.

In one embodiment of the present invention each of the internucleosidelinking groups of the antisense oligomeric compounds and the senseoligomeric compounds is a phosphorus containing internucleoside linkage.In a preferred embodiment each of the internucleoside linking groups isindependently a phosphodiester or a phosphorothioate internucleosidelinkage. In another preferred embodiment each of the internucleosidelinking groups is phosphodiester internucleoside linkage. In a furtherpreferred embodiment each of the internucleoside linking groups is aphosphorothioate internucleoside linkage.

In one embodiment of the present invention each of the antisenseoligomeric compounds and the sense oligomeric compounds optionallycomprise a terminal phosphate group, a terminal stabilizing or cappinggroup, a 3′-overhang or a conjugate group. In one preferred embodimentthe 3′-terminus of the antisense oligomeric compound comprises astabilizing or conjugate group wherein a preferred stabilizing group isa dTdT dimer. In another embodiment the 5′-terminus of the antisenseoligomeric compound comprises a 5′-phosphate group.

In one embodiment of the present invention the the antisense oligomericcompound comprises at least one terminal cap moiety. In a preferredembodiment a terminal cap moiety is attached to one or both of the3′-terminal and 5′-terminal ends of the antisense oligomeric compound. Apreferred terminal cap moiety includes an inverted deoxy abasic moiety.

In one embodiment of the present invention the the antisense oligomericcompound has from about 8 to about 80 nucleobases in length. In afurther embodiment the antisense oligomeric compound has from about 12to about 50 nucleobases in length. In an even further embodiment theantisense oligomeric compound has from about 15 to about 30 nucleobasesin length.

In one embodiment of the present invention each of the sense oligomericcompounds has from about 2 to about 12 nucleobases in length. In apreferred embodiment each of the sense oligomeric compounds has fromabout 2 to about 10 nucleobases in length. In another preferredembodiment each of the sense oligomeric compounds has from about 2 toabout 7 nucleobases in length.

In one embodiment of the present invention is a method of inhibitinggene expression comprising contacting one or more cells, a tissue or ananimal with a composition of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising an antisenseoligomeric compound and at least one sense oligomeric compoundhybridized thereto. The sense oligomeric compounds are shorter than theantisense oligomeric compounds and hybridize to the antisense oligomericcompounds forming double stranded regions. The antisense oligomericcompound can have multiple sense strands bound thereto forming onecontinuous double stranded composition or can have any configurationresulting from one or more double stranded regions and one or moresingle stranded regions. At least a portion of the antisense oligomericcompound is complementary to and hybridizes with a nucleic acid target.The compositions can comprise a single strand with the complementarysense strands covalently linked to the antisense oligomeric compounds.

In one embodiment short sense oligomeric compounds having at leastpartial complementarity to the antisense oligomeric compound areconnected thereby forming a single strand. The connection can be aregion of non-hybridizing nucleobases or a bifunctional tethering groupe.g. a non-nucleic acid linker (see for example WO 94/01550). Examplesof such linkers include substituted or unsubstituted alkyl groups. Apreferred linking group amenable to the present invention is a polyethylene glycol linker with (ethylene glycol)₁₋₆ being a preferredrange. The synthesis can be easily caried out using commerciallyavailable triethylene glycol that has been functionalized to have adimethoxytrityl (or dimethyltrityl) protective group at one end and acyanoethylphosphoramidite group at the other end.

In one aspect of the present invention one or more complementary sensestrands are bound to an antisense strand that has complementary to anucleic acid target. Although the antisense oligomeric compounds can befrom 8 to 80 nucleobases in length, antisense oligomeric compounds canbe from about 12 to about 50 nucleobases in length or about 15 to about30 nucleobases in length.

In one aspect of the present invention the sense oligomeric compoundsare shorter than the antisense oligomeric compounds and have at least aregion that is complementary to and hybridizes to the antisenseoligomeric comounds. The sense oligomeric compounds can be 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12 nucleobases in length. In one aspect there isone sense oligomeric compound that is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or12 nucleobases in length hybridized at least partially and preferrablycompletely to the antisense oligomeric compound. In another aspect thereare two sense oligomeric compounds that are each 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or about 12 nucleobases in length hybridized at least partiallyto the antisense oligomeric compound. In another aspect there are 3 ormore small sense oligomeric compounds that are each 2, 3, 4, 5, 6 orabout 7 nucleobases in length hybridized at least partially to theantisense oligomeric compound.

The compositions of the present invention can comprise antisenseoligomeric compounds and sense oligomeric compounds that are naturallyoccurring or chemically modified to give a plurality of motifs for eachsequence. One preferred modification of the antisense oligomericcompound is a 5′-phosphate group. The bases, sugars and internucleosidelinkages of oligonucleotides comprising the compositions of the presentinvention can be prepared with a myriad of chemical modifications asshown herein and as is know in the art.

Compositions of the present invention will be useful for the modulationof gene expression. In one aspect of the present invention a targetedcell, group of cells, a tissue or an animal is contacted with acomposition of the invention to effect reduction, accumulation orincreased specificity by reduction of off-target effects of mRNA or RNAthat can directly inhibit gene expression. In another embodiment thereduction of mRNA or RNA indirectly upregulates a non-targeted genethrough a pathway that relates the targeted gene to a non-targeted gene.Methods and models for the regulation of genes using oligomericcompounds of the invention are illustrated in the examples.

In another embodiment, a method of inhibiting gene expression isdisclosed comprising contacting one or more cells, a tissue or an animalwith a composition of the invention. Numerous procedures of how to usethe compositions of the present invention are illustrated in theexamples section.

Compositions of the invention modulate gene expression by hybridizing toa nucleic acid target resulting in loss of its normal function. As usedherein, the term “target nucleic acid” or “nucleic acid target” is usedfor convenience to encompass any nucleic acid capable of being targetedincluding without limitation DNA, RNA (including pre-mRNA and mRNA orportions thereof) transcribed from such DNA, and also cDNA derived fromsuch RNA. In one embodiment of the invention the target nucleic acid isa messenger RNA. In a further embodiment the degradation of the targetedmessenger RNA is facilitated by a RISC complex that is formed witholigomeric compounds of the invention. As such, the compounds of theinvention are known as optimized “RISC loaders.” In another embodimentthe degradation of the targeted messenger RNA is facilitated by anuclease such as RNase III. In another embodiment, degradation of targetmRNA or target RNA is by the nuclease, RNAse III. The degradationelicited by the compounds of the invention may be in the cytoplasm ornucleus of the cell.

The hybridization of an oligomeric compound of this invention with itstarget nucleic acid is generally referred to as “antisense”.Consequently, the mechanism in the practice of the embodiments of theinvention is referred to herein as “antisense inhibition.” Suchantisense inhibition is typically based upon hydrogen bonding-basedhybridization of oligonucleotide strands or segments such that at leastone strand or segment is cleaved, degraded, or otherwise renderedinoperable. In this regard, it is presently preferred to target specificnucleic acid molecules and their functions for such antisenseinhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. In the context of the present invention,“modulation” and “modulation of expression” mean either an increase(stimulation) or a decrease (inhibition) in the amount or levels of anucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition isoften the preferred form of modulation of expression and mRNA is often apreferred target nucleic acid.

The compositions and methods of the present invention are also useful inthe study, characterization, validation and modulation of smallnon-coding RNAs. These include, but are not limited to, microRNAs(miRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA),small temporal RNAs (stRNA) and tiny non-coding RNAs (tncRNA) or theirprecursors or processed transcripts or their association with othercellular components.

Small non-coding RNAs have been shown to function in variousdevelopmental and regulatory pathways in a wide range of organisms,including plants, nematodes and mammals. MicroRNAs are small non-codingRNAs that are processed from larger precursors by enzymatic cleavage andinhibit translation of mRNAs. stRNAs, while processed from precursorsmuch like miRNAs, have been shown to be involved in developmental timingregulation. Other non-coding small RNAs are involved in events asdiverse as cellular splicing of transcripts, translation, transport, andchromosome organization.

As modulators of small non-coding RNA function, the compositions of thepresent invention find utility in the control and manipulation ofcellular functions or processes such as regulation of splicing,chromosome packaging or methylation, control of developmental timingevents, increase or decrease of target RNA expression levels dependingon the timing of delivery into the specific biological pathway andtranslational or transcriptional control. In addition, the compositionsof the present invention can be modified in order to optimize theireffects in certain cellular compartments, such as the cytoplasm,nucleus, nucleolus or mitochondria.

The compositions of the present invention can further be used toidentify components of regulatory pathways of RNA processing ormetabolism as well as in screening assays or devices.

The compounds and compositions of the present invention can further beused to minimize off target effects.

Oligomeric Compounds

In the context of the present invention, the term “oligomeric compound”refers to a polymeric structure capable of hybridizing a region of anucleic acid molecule. This term includes oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics andcombinations of these. Oligomeric compounds are routinely preparedlinearly but can be joined or otherwise prepared to be circular and mayalso include branching. Oligomeric compounds can included doublestranded constructs such as for example two strands hybridized to formdouble stranded compounds. The double stranded compounds can be linkedor separate and can include overhangs on the ends. In general anoligomeric compound comprises a backbone of linked momeric subunitswhere each linked momeric subunit is directly or indirectly attached toa heterocyclic base moiety. Oligomeric compounds may also includemonomeric subunits that are not linked to a heterocyclic base moietythereby providing abasic sites. The linkages joining the monomericsubunits, the sugar moieties or surrogates and the heterocyclic basemoieties can be independently modified giving rise to a plurality ofmotifs for the resulting oligomeric compounds including hemimers,gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be joined to form a circular structure by hybridization orby formation of a covalent bond, however, open linear structures aregenerally preferred. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside linkagesof the oligonucleotide. The normal internucleoside linkage of RNA andDNA is a 3′ to 5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA). This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleosidelinkages. The term “oligonucleotide analog” refers to oligonucleotidesthat have one or more non-naturally occurring portions which function ina similar manner to oligonulceotides. Such non-naturally occurringoligonucleotides are often preferred the naturally occurring formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In the context of this invention, the term “oligonucleoside” refers to asequence of nucleosides that are joined by internucleoside linkages thatdo not have phosphorus atoms. Internucleoside linkages of this typeinclude short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixedheteroatom cycloalkyl, one or more short chain heteroatomic and one ormore short chain heterocyclic. These internucleoside linkages includebut are not limited to siloxane, sulfide, sulfoxide, sulfone, acetyl,formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl,alkeneyl, sulfamate; methyleneimino, methylenehydrazino, sulfonate,sulfonamide, amide and others having mixed N, O, S and CH₂ componentparts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Further included in the present invention are oligomeric compounds suchas antisense oligomeric compounds, antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, alternatesplicers, primers, probes, and other oligomeric compounds whichhybridize to at least a portion of the target nucleic acid. As such,these oligomeric compounds may be introduced in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the oligomericcompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense oligomeric compoundswhich are “DNA-like” elicit RNAse H. Activation of RNase H, therefore,results in cleavage of the RNA target, thereby greatly enhancing theefficiency of oligonucleotide-mediated inhibition of gene expression.Similar roles have been postulated for other ribonucleases such as thosein the RNase III and ribonuclease L family of enzymes.

While the preferred form of antisense oligomeric compound is asingle-stranded antisense oligonucleotide, in many species theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

As optimized RISC loaders, the compounds of the present inventionpreferentially bind to dsRNA binding motifs in cellular complexes. Onesuch complex is the RISC (RNA induced silencing complex) complex.

In addition to the modifications described above, the nucleosides of theoligomeric compounds of the invention can have a variety of othermodification so long as these other modifications either alone or incombination with other nucleosides enhance one or more of the desiredproperties described above. Thus, for nucleotides that are incorporatedinto oligonucleotides of the invention, these nucleotides can have sugarportions that correspond to naturally-occurring sugars or modifiedsugars. Representative modified sugars include carbocyclic or acyclicsugars, sugars having substituent groups at one or more of their 2′, 3′or 4′ positions and sugars having substituents in place of one or morehydrogen atoms of the sugar. Additional nucleosides amenable to thepresent invention having altered base moieties and or altered sugarmoieties are disclosed in U.S. Pat. No. 3,687,808 and PCT applicationPCT/US89/02323.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigonucleotides are best described as being structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic wild type oligonucleotides. All sucholigonucleotides are comprehended by this invention so long as theyfunction effectively to mimic the structure of a desired RNA or DNAstrand. A class of representative base modifications include tricycliccytosine analog, termed “G clamp” (Lin, et al, J. Am. Chem. Soc. 1998,120, 8531). This analog makes four hydrogen bonds to a complementaryguanine (G) within a helix by simultaneously recognizing theWatson-Crick and Hoogsteen faces of the targeted G. This G clampmodification when incorporated into phosphorothioate oligonucleotides,dramatically enhances antisense potencies in cell culture. Theoligonucleotides of the invention also can includephenoxazine-substituted bases of the type disclosed by Flanagan, et al.,Nat. Biotechnol. 1999, 17(1), 48-52.

The antisense oligomeric compounds in accordance with this inventionpreferably comprise from about 8 to about 80 nucleobases (i.e. fromabout 8 to about 80 linked nucleosides). One of ordinary skill in theart will appreciate that the invention embodies oligomeric compounds of8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,or 80 nucleobases in length.

In one preferred embodiment, the antisense oligomeric compounds of theinvention are 12 to 50 nucleobases in length. One having ordinary skillin the art will appreciate that this embodies oligomeric compounds of12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, or 50 nucleobases in length.

In another preferred embodiment, the antisense oligomeric compounds ofthe invention are 15 to 30 nucleobases in length. One having ordinaryskill in the art will appreciate that this embodies oligomeric compoundsof 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleobases in length.

Particularly preferred antisense oligomeric compounds areoligonucleotides from about 12 to about 50 nucleobases, even morepreferably those comprising from about 15 to about 30 nucleobases.

Oligomer and Monomer Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside linkage or in conjunctionwith the sugar ring the backbone of the oligonucleotide. The normalinternucleoside linkage that makes up the backbone of RNA and DNA is a3′ to 5′ phosphodiester linkage.

Chimeric oligomeric compounds

It is not necessary for all positions in a oligomeric compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligomeric compound oreven at a single monomeric subunit such as a nucleoside within aoligomeric compound. The present invention also includes oligomericcompounds which are chimeric oligomeric compounds. “Chimeric” oligomericcompounds or “chimeras,” in the context of this invention, areoligomeric compounds containing two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Such oligomeric compounds have also been referred to in the artas hybrids hemimers, gapmers or inverted gapmers. Representative UnitedStates patents that teach the preparation of such hybrid structuresinclude, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797;5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

Oligomer Mimetics

Another preferred group of oligomeric compounds amenable to the presentinvention includes oligonucleotide mimetics. The term mimetic as it isapplied to oligonucleotides is intended to include oligomeric compoundswherein the furanose ring or the furanose ring and the internucleotidelinkage are replaced with novel groups, replacement of only the furanosering is also referred to in the art as being a sugar surrogate. Theheterocyclic base moiety or a modified heterocyclic base moiety ismaintained for hybridization with an appropriate target nucleic acid.

One such oligomeric compound, an oligonucleotide mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). PNA's have favorable hybridizationproperties, high biological stability and are electrostatically neutralmolecules. In one recent study PNA's were used to correct aberrantsplicing in a transgenic mouse model (Sazani et al., Nat. Biotechnol.,2002, 20, 1228-1233). In PNA oligomeric compounds, the sugar-backbone ofan oligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA oligomeric compounds include, but are not limitedto, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which isherein incorporated by reference. PNA's can be obtained commerciallyfrom Applied Biosystems (Foster City, Calif., USA).

Numerous modifications have been made to the basic PNA backbone since itwas introduced in 1991 by Nielsen and coworkers (Nielsen et al.,Science, 1991, 254, 1497-1500). The basic structure is shown below:

wherein

-   -   Bx is a heterocyclic base moiety;    -   T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted        or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted        C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl,        alkylsulfonyl, arylsulfonyl, a chemical functional group, a        reporter group, a conjugate group, a D or L α-amino acid linked        via the α-carboxyl group or optionally through the ω-carboxyl        group when the amino acid is aspartic acid or glutamic acid or a        peptide derived from D, L or mixed D and L amino acids linked        through a carboxyl group, wherein the substituent groups are        selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl,        nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and        alkynyl;    -   T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the        α-amino group or optionally through the ω-amino group when the        amino acid is lysine or ornithine or a peptide derived from D, L        or mixed D and L amino acids linked through an amino group, a        chemical functional group, a reporter group or a conjugate        group;    -   Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;    -   Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,        —C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the        α-carboxyl group or optionally through the ω-carboxyl group when        the amino acid is aspartic acid or glutamic acid or a peptide        derived from D, L or mixed D and L amino acids linked through a        carboxyl group;    -   Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl,        —C(═O)—CH₃, benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;    -   each J is O, S or NH;    -   R₅ is a carbonyl protecting group; and    -   n is from 2 to about 50.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. A preferred class of linking groups have been selected togive a non-ionic oligomeric compound. The non-ionic morpholino-basedoligomeric compounds are less likely to have undesired interactions withcellular proteins. Morpholino-based oligomeric compounds are non-ionicmimics of oligonucleotides which are less likely to form undesiredinteractions with cellular proteins (Dwaine A. Braasch and David R.Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-basedoligomeric compounds have been studied in ebrafish embryos (see:Genesis, volume 30, issue 3, 2001 and Heasman, J., Dev. Biol., 2002,243, 209-214). Further studies of Morpholino-based oligomeric compoundshave also been reported (see: Nasevicius et al., Nat Genet., 2000, 26,216-220; and Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97,9591-9596). Morpholino-based oligomeric compounds are disclosed in U.S.Pat. No. 5,034,506, issued Jul. 23, 1991. The morpholino class ofoligomeric compounds have been prepared having a variety of differentlinking groups joining the monomeric subunits.

Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

wherein

-   -   T₁ is hydroxyl or a protected hydroxyl;    -   T₅ is hydrogen or a phosphate or phosphate derivative;    -   L₂ is a linking group; and    -   n is from 2 to about 50.

A further class of oligonucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present inan DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Ingeneral the incorporation of CeNA monomers into a DNA chain increasesits stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes. The study of incorporating CeNA structures into naturalnucleic acid structures was shown by NMR and circular dichroism toproceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. Coli RNase resulting in cleavage of the targetRNA strand.

The general formula of CeNA is shown below:

wherein

-   -   each Bx is a heterocyclic base moiety;    -   T₁ is hydroxyl or a protected hydroxyl; and    -   T2 is hydroxyl or a protected hydroxyl.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid)can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

A further preferred modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugarring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens.Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; andOrum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243). The linkage ispreferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun.,1998, 4, 455-456). LNA and LNA analogs display very high duplex thermalstabilities with complementary DNA and RNA (Tm=+3 to +10 C), stabilitytowards 3′-exonucleolytic degradation and good solubility properties.LNA's are commercially available from ProLigo (Paris, France andBoulder, Colo., USA). The basic structure of LNA showing the bicyclicring system is shown below:

Another isomer of LNA that has also been studied is α-L-LNA which hasbeen shown to have superior stability against a 3′-exonuclease (Friedenet al., Nucleic Acids Research, 2003, 21, 6365-6372). The α-L-LNA's wereincorporated into antisense gapmers and chimeras that showed potentantisense activity. The structure of α-L-LNA is shown below:

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (N) examination of anLNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.DNA•LNA chimeras have been shown to efficiently inhibit gene expressionwhen targeted to a variety of regions (5′-untranslated region, region ofthe start codon or coding region) within the luciferase mRNA (Braasch etal., Nucleic Acids Research, 2002, 30, 5160-5167).

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad Sci. U.S.A., 2000, 97,5633-5638.) The authors have demonstrated that LNAs confer severaldesired properties to antisense agents. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity in assay systems as disparate asG-protein-coupled receptor signaling in living rat brain and detectionof reporter genes in Escherichia coli. Lipofectin-mediated efficientdelivery of LNA into living human breast cancer cells has also beenaccomplished. Further successful in vivo studies involving LNA's haveshown knock-down of the rat delta opioid receptor without toxicity(Wahlestedt et al., Proc. Natl. Acad. Sci., 2000, 97, 5633-5638) and inanother study showed a blockage of the translation of the large subunitof RNA polymerase II (Fluiter et al., Nucleic Acids Res., 2003, 31,953-962).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Another oligonucleotide mimetic amenable to the present invention thathas been prepared and studied is threose nucleic acid. Thisoligonucleotide mimetic is based on threose nucleosides instead ofribose nucleosides and has the general structure shown below:

Initial interest in (3′,2′)-α-L-threose nucleic acid (TNA) was directedto the question of whether a DNA polymerase existed that would copy theTNA. It was found that certain DNA polymerases are able to copy limitedstretches of a TNA template (reported in C&EN/Jan. 13, 2003).

In another study it was determined that TNA is capable of antiparallelWatson-Crick base pairing with complementary DNA, RNA and TNAoligonucleotides (Chaput et al., J. Am. Chem. Soc., 2003, 125, 856-857).

In one study (3′,2′)-α-L-threose nucleic acid was prepared and comparedto the 2′ and 3′ amidate analogs (Wu et al., Organic Letters, 2002,4(8), 1279-1282). The amidate analogs were shown to bind to RNA and DNAwith comparable strength to that of RNA/DNA.

Further oligonucleotide mimetics have been prepared to incude bicyclicand tricyclic nucleoside analogs having the formulas (amidite monomersshown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; Renneberg et al., J. Am.Chem. Soc., 2002, 124, 5993-6002; and Renneberg et al., Nucleic acidsres., 2002, 30, 2751-2757). These modified nucleoside analogs have beenoligomerized using the phosphoramidite approach and the resultingoligomeric compounds containing tricyclic nucleoside analogs have shownincreased thermal stabilities (Tm's) when hybridized to DNA, RNA anditself Oligomeric compounds containing bicyclic nucleoside analogs haveshown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids which incorporate a phosphorus group inthe backbone. This class of olignucleotide mimetic is reported to haveuseful physical and biological and pharmacological properties in theareas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat.Nos. 5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Further oligonucleotide mimetics amenable to the present invention havebeen prepared wherein a cyclobutyl ring replaces the naturally occurringfuranosyl ring.

Modified Internucleoside Linkages

Specific examples of preferred antisense oligomeric compounds useful inthis invention include oligonucleotides containing modified e.g.non-naturally occurring internucleoside linkages. As defined in thisspecification, oligonucleotides having modified internucleoside linkagesinclude internucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage(phosphorothioate) did not significantly interfere with RNAi activity.Based on this observation, it is suggested that certain preferredoligomeric compounds of the invention can also have one or more modifiedinternucleoside linkages. A preferred phosphorus containing modifiedinternucleoside linkage is the phosphorothioate internucleoside linkage.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, phosphonoacetateand thiophosphonoacetate, selenophosphates and boranophosphates havingnormal 3′-5′ linkages, 2′-5′ linked analogs of these, and those havinginverted polarity wherein one or more internucleotide linkages is a 3′to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides havinginverted polarity comprise a single 3′ to 3′ linkage at the 3′-mostinternucleotide linkage i.e. a single inverted nucleoside residue whichmay be abasic (the nucleobase is missing or has a hydroxyl group inplace thereof). Various salts, mixed salts and free acid forms are alsoincluded.

N3′-P5′-phosphoramidates have been reported to exhibit both a highaffinity towards a complementary RNA strand and nuclease resistance(Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144).N3′-P5′-phosphoramidates have been studied with some success in vivo tospecifically down regulate the expression of the c-myc gene (Skorski etal., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat.Biotechnol., 2001, 19, 40-44).

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

In further embodiments of the invention, oligomeric compounds have oneor more phosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂]. The MMI type internucleoside linkages are disclosedin the above referenced U.S. Pat. No. 5,489,677. Preferred amideinternucleoside linkages are disclosed in the above referenced U.S. Pat.No. 5,602,240.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugars

Oligomeric compounds of the invention may also contain one or moresubstituted sugar moieties. Preferred oligomeric compounds comprise asugar substituent group selected from: OH; F; O—, S—, or N-alkyl; O—,S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise a sugarsubstituent group selected from: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH2OCH₂N(CH₃)₂.

Other preferred sugar substituent groups include methoxy (—O—CH₃),aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F (see: Loc et al., Biochemistry, 2002,41, 3457-3467). Similar modifications may also be made at otherpositions on the oligomeric compoiund, particularly the 3′ position ofthe sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

Further representative sugar substituent groups include groups offormula I_(a) or II_(a):

wherein:

-   -   R_(b) is O, S or NH;    -   R_(d) is a single bond, O, S or C(═O);    -   R_(e) is C₁-C₁₀ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),        N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);    -   R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;    -   R_(r) is —R_(x)—R_(y);    -   each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,        C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl,        substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or        unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a        chemical functional group or a conjugate group, wherein the        substituent groups are selected from hydroxyl, amino, alkoxy,        carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,        alkyl, aryl, alkenyl and alkynyl;    -   or optionally, R_(u) and R_(v), together form a phthalimido        moiety with the nitrogen atom to which they are attached;    -   each R_(w) is, independently, substituted or unsubstituted        C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);    -   R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);    -   R_(x) is a bond or a linking moiety;    -   R_(y) is a chemical functional group, a conjugate group or a        solid support medium;    -   each R_(m) and R_(n) is, independently, H, a nitrogen protecting        group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, wherein the substituent groups are selected from        hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,        thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺,        N(R_(u))(R_(v)), guanidino and acyl where said acyl is an acid        amide or an ester;    -   or R_(m) and R_(n), together, are a nitrogen protecting group,        are joined in a ring structure that optionally includes an        additional heteroatom selected from N and O or are a chemical        functional group;    -   R_(i) is OR_(z), SR_(z), or N(R_(z))₂;    -   each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);    -   R_(f), R_(g) and R_(h) comprise a ring system having from about        4 to about 7 carbon atoms or having from about 3 to about 6        carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are        selected from oxygen, nitrogen and sulfur and wherein said ring        system is aliphatic, unsaturated aliphatic, aromatic, or        saturated or unsaturated heterocyclic;    -   R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,        alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to        about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,        N(R_(k))(R_(m)) OR_(k), halo, SR_(k) or CN;    -   m_(a) is 1 to about 10;    -   each mb is, independently, 0 or 1;    -   mc is 0 or an integer from 1 to 10;    -   md is an integer from 1 to 10;    -   me is from 0, 1 or 2; and    -   provided that when mc is 0, md is greater than 1.

Representative substituents groups of Formula I are disclosed in U.S.patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled“Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by referencein its entirety.

Representative cyclic substituent groups of Formula II are disclosed inU.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998,entitled “RNA Targeted 2′-Oligomeric compounds that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Particularly preferred sugar substituent groups includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10.

Representative guanidino substituent groups that are shown in formulaIII and IV are disclosed in co-owned U.S. patent application Ser. No.09/349,040, entitled “Functionalized Oligomers”, filed Jul. 7, 1999,hereby incorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

Modified Nucleobases/Naturally occurring Nucleobases

Oligomeric compounds may also include nucleobase (often referred to inthe art simply as “base” or “heterocyclic base moiety”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesalso referred herein as heterocyclic base moieties include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Oligomeric compounds of the present invention can also includepolycyclic heterocyclic compounds in place of one or more heterocyclicbase moieties. A number of tricyclic heterocyclic comounds have beenpreviously reported. These compounds are routinely used in antisenseapplications to increase the binding properties of the modified strandto a target strand. The most studied modifications are targeted toguanosines hence they have been termed G-clamps or cytidine analogs.Many of these polycyclic heterocyclic compounds have the generalformula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one(R_(10═)O, R₁₁—R₁₄═H) [Kurchavov, et al., Nucleosides and nucleotides,1997, 16, 1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁—R₁₄═H),[Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁—R₁₄═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions(also see U.S. patentapplication entitled “Modified Peptide Nucleic Acids” filed May 24,2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are commonly owned with this applicationand are herein incorporated by reference in their entirety).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R_(10═)O, R₁₁═—O—(CH₂)₂—NH₂,R₁₂₋₁₄═H ) [Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120,8531-8532]. Binding studies demonstrated that a single incorporationcould enhance the binding affinity of a model oligonucleotide to itscomplementary target DNA or RNA with a ΔT_(m) of up to 18° relative to5-methyl cytosine (dC5^(me)), which is the highest known affinityenhancement for a single modification, yet. On the other hand, the gainin helical stability does not compromise the specificity of theoligonucleotides. The T_(m) data indicate an even greater discriminationbetween the perfect match and mismatched sequences compared to dC5^(me).It was suggested that the tethered amino group serves as an additionalhydrogen bond donor to interact with the Hoogsteen face, namely the O6,of a complementary guanine thereby forming 4 hydrogen bonds. This meansthat the increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992,which issued on Dec. 28, 1999, the contents of both are commonlyassigned with this application and are incorporated herein in theirentirety.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity [Lin, K-Y; Matteucci, M. J.Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement was evenmore pronounced in case of G-clamp, as a single substitution was shownto significantly improve the in vitro potency of a 20 mer2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimizeoligonucleotide design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further modified polycyclic heterocyclic compounds useful asheterocyclcic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patentapplication Ser. No. 09/996,292 filed Nov. 28, 2001, certain of whichare commonly owned with the instant application, and each of which isherein incorporated by reference.

Conjugates

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more moieties or conjugates forenhancing the activity, cellular distribution or cellular uptake of theresulting oligomeric compounds. In one embodiment such modifiedoligomeric compounds are prepared by covalently attaching conjugategroups to functional groups such as hydroxyl or amino groups. Conjugategroups of the invention include intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugatesgroups include cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamicproperties, in the context of this invention, include groups thatimprove oligomer uptake, enhance oligomer resistance to degradation,and/or strengthen sequence-specific hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Representative conjugate groups are disclosedin International Patent Application PCT/US92/09196, filed Oct. 23, 1992the entire disclosure of which is incorporated herein by reference.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of oligomeric compounds toenhance properties such as for example nuclease stability. Included instabilizing groups are cap structures. By “cap structure or terminal capmoiety” is meant chemical modifications, which have been incorporated ateither terminus of oligonucleotides (see for example Wincott et al., WO97/26270, incorporated by reference herein). These terminalmodifications protect the oligomeric compounds having terminal nucleicacid molecules from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present onboth termini. In non-limiting examples, the 5′-cap includes invertedabasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl)nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270,incorporated by reference herein).

Further 3′-cap structures of the present invention include, for example4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide;4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexylphosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modifiedbase nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide;3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety;5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate;1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridgingor non bridging methylphosphonate and 5′-mercapto moieties (for moredetails see Beaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporatedby reference herein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in WO 03/004602 published on Jan. 16, 2003.

3′-Endo Modifications

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but aren'tlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric triggers of RNAi having one or morenucleosides modified in such a way as to favor a C3′-endo typeconformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element, as illustrated in FIG. 2, below (Gallo et al.,Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem.,(1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,747-754.) Alternatively, preference for the 3′-endo conformation can beachieved by deletion of the 2′-OH as exemplified by2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem.(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett.(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation. Along similar lines, oligomeric triggers of RNAi responsemight be composed of one or more nucleosides modified in such a way thatconformation is locked into a C3′-endo type conformation, i.e. LockedNucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), andethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic &Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modifiednucleosides amenable to the present invention are shown below in TableI. These examples are meant to be representative and not exhaustive.TABLE I

The preferred conformation of modified nucleosides and their oligomerscan be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligonucleotides of the presentinvention. The synthesis of numerous of the modified nucleosidesamenable to the present invention are known in the art (see for example,Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend,1988, Plenum press., and the examples section below.)

In one aspect, the present invention is directed to oligomers that areprepared having enhanced properties compared to native RNA againstnucleic acid targets. A target is identified and an oligomer is selectedhaving an effective length and sequence that is complementary to aportion of the target sequence. Each nucleoside of the selected sequenceis scrutinized for possible enhancing modifications. A preferredmodification would be the replacement of one or more RNA nucleosideswith nucleosides that have the same 3′-endo conformational geometry.Such modifications can enhance chemical and nuclease stability relativeto native RNA while at the same time being much cheaper and easier tosynthesize and/or incorporate into an oligonucleotide. The selectedsequence can be further divided into regions and the nucleosides of eachregion evaluated for enhancing modifications that can be the result of achimeric configuration. Consideration is also given to the 5′ and3′-termini as there are often advantageous modifications that can bemade to one or more of the terminal nucleosides. The oligomericcompounds of the present invention include at least one 5′-modifiedphosphate group on a single strand or on at least one 5′-position of adouble stranded sequence or sequences. Further modifications are alsoconsidered such as internucleoside linkages, conjugate groups,substitute sugars or bases, substitution of one or more nucleosides withnucleoside mimetics and any other modification that can enhance theselected sequence for its intended target.

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (Tm's)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and O4′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a O4′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as but not limitedto antisense and RNA interference as these mechanisms require thebinding of a synthetic oligomer strand to an RNA target strand. In thecase of antisense, effective inhibition of the mRNA requires that theantisense DNA have a very high binding affinity with the mRNA. Otherwisethe desired interaction between the synthetic oligomer strand and targetmRNA strand will occur infrequently, resulting in decreased efficacy.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2-methoxyethoxy(2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000). One of the immediate advantages of the 2′-MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl. Oligomers having the 2′-O-methoxyethyl substituent alsohave been shown to be antisense inhibitors of gene expression withpromising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995,78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligomers havingthe 2′-MOE modification displayed improved RNA affinity and highernuclease resistance. Chimeric oligomers having 2′-MOE substituents inthe wing nucleosides and an internal region of deoxy-phosphorothioatenucleotides (also termed a gapped oligomer or gapmer) have showneffective reduction in the growth of tumors in animal models at lowdoses. 2′-MOE substituted oligomers have also shown outstanding promiseas antisense agents in several disease states. One such MOE substitutedoligomer is presently being investigated in clinical trials for thetreatment of CMV retinitis.

Chemistries Defined

Unless otherwise defined herein, alkyl means C₁-C₁₂, preferably C₁-C₈,and more preferably C₁-C₆, straight or (where possible) branched chainaliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, preferablyC₁-C₈, and more preferably C₁-C₆, straight or (where possible) branchedchain aliphatic hydrocarbyl containing at least one, and preferablyabout 1 to about 3, hetero atoms in the chain, including the terminalportion of the chain. Preferred heteroatoms include N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, preferablyC₃-C₈, and more preferably C₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, preferably C₂-C₈,and more preferably C₂-C₆ alkenyl, which may be straight or (wherepossible) branched hydrocarbyl moiety, which contains at least onecarbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, preferably C₂-C₈,and more preferably C₂-C₆ alkynyl, which may be straight or (wherepossible) branched hydrocarbyl moiety, which contains at least onecarbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moietycontaining at least three ring members, at least one of which is carbon,and of which 1, 2 or three ring members are other than carbon.Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred heterocycloalkyl groupsinclude morpholino, thiomorpholino, piperidinyl, piperazinyl,homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino,pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl,tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl,furanyl, pyranyl, and tetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Preferred aryl rings haveabout 6 to about 20 ring carbons. Especially preferred aryl ringsinclude phenyl, napthyl, anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containingat least one fully unsaturated ring, the ring consisting of carbon andnon-carbon atoms. Preferably the ring system contains about 1 to about 4rings. Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred hetaryl moieties includepyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl,pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl,benzothiophenyl, etc.

Unless otherwise defined herein, where a moiety is defined as a compoundmoiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl andalkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is agroup, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have theirordinary meanings. Preferred halo (halogen) substituents are Cl, Br, andI.

The aforementioned optional substituents are, unless otherwise hereindefined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc.

In all the preceding formulae, the squiggle (−) indicates a bond to anoxygen or sulfur of the 5′-phosphate. Phosphate protecting groupsinclude those described in U.S. Pat. No. 5,760,209, U.S. Pat. No.5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat.No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S.Pat. No. 6,465,628 each of which is expressly incorporated herein byreference in its entirety.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides is performedaccording to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA:Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesisas appropriate. In addition specific protocols for the synthesis ofoligomeric compounds of the invention are illustrated in the examplesbelow.

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The present invention is also useful for the preparation of oligomericcompounds incorporating at least one 2′-O-protected nucleoside. Afterincorporation and appropriate deprotection the 2′-O-protected nucleosidewill be converted to a ribonucleoside at the position of incorporation.The number and position of the 2-ribonucleoside units in the finaloligomeric compound can vary from one at any site or the strategy can beused to prepare up to a full 2′-OH modified oligomeric compound. All2′-O-protecting groups amenable to the synthesis of oligomeric compoundsare included in the present invention. In general a protected nucleosideis attached to a solid support by for example a succinate linker. Thenthe oligonucleotide is elongated by repeated cycles of deprotecting the5′-terminal hydroxyl group, coupling of a further nucleoside unit,capping and oxidation (alternatively sulfurization). In a morefrequently used method of synthesis the completed oligonucleotide iscleaved from the solid support with the removal of phosphate protectinggroups and exocyclic amino protecting groups by treatment with anammonia solution. Then a further deprotection step is normally requiredfor removal of the more specialized protecting groups used for theprotection of 2′-hydroxyl groups thereby affording the fully deprotectedoligonucleotide.

A large number of 2′-O-protecting groups have been used for thesynthesis of oligoribonucleotides but over the years more effectivegroups have been discovered. The key to an effective 2′-O-protectinggroup is that it is capable of selectively being introduced at the2′-O-position and that it can be removed easily after synthesis withoutthe formation of unwanted side products. The protecting group also needsto be inert to the normal deprotecting, coupling, and capping stepsrequired for oligoribonucleotide synthesis. Some of the protectinggroups used initially for oligoribonucleotide synthesis includedtetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groupsare not compatible with all 5′-O-protecting groups so modified versionswere used with 5′-DMT groups such as1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has identifieda number of piperidine derivatives (like Fpmp) that are useful in thesynthesis of oligoribonucleotides including1-[(chloro-4-methyl)phenyl]-4′-methoxypiperidin-4-yl (Reese et al.,Tetrahedron Lett., 1986, (27), 2291). Another approach was to replacethe standard 5′-DMT (dimethoxytrityl) group with protecting groups thatwere removed under non-acidic conditions such as levulinyl and9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile2′-protecting groups for oligoribonucleotide synthesis. Another morewidely used protecting group initially used for the synthesis ofoligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al.,Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett.,1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I., 2762). The2′-O-protecting groups can require special reagents for their removalsuch as for example the t-butyldimethylsilyl group is normally removedafter all other cleaving/deprotecting steps by treatment of theoligomeric compound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2′-O-protecting groups(Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoridelabile and photolabile protecting groups that are removed using moderateconditions. One photolabile group that was examined was the[2-(nitrobenzyl)oxy]methyl (nbm) protecting group (Schwartz et al.,Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examinedincluded a number structurally related formaldehyde acetal-derived,2′-O-protecting groups. Also prepared were a number of relatedprotecting groups for preparing 2′-O-alkylated nucleosidephosphoramidites including 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O—CH₂—O—Si(iPr)₃, TOM). One 2′-O-protecting group that was preparedto be used orthogonally to the TOM group was2′-O—[(R)-1-(2-nitrophenyl)ethyloxy)methyl] ((R)-mnbm).

Another strategy using a fluoride labile 5′-O-protecting group (non-acidlabile) and an acid labile 2′-O-protecting group has been reported(Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number ofpossible silyl ethers were examined for 5′-O-protection and a number ofacetals and orthoesters were examined for 2′-O-protection. Theprotection scheme that gave the best results was 5′-O-silyl ether-2′-ACE(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether(DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses amodified phosphoramidite synthesis approach in that some differentreagents are required that are not routinely used for RNA/DNA synthesis.

Although a lot of research has focused on the synthesis ofoligoribonucleotides the main RNA synthesis strategies that arepresently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS),5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),2′-O-[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM), and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. Such an activator would also be amenable to the presentinvention.

The primary groups being used for commercial RNA synthesis are:

-   -   TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;    -   TOM=2′-O-[(triisopropylsilyl)oxy]methyl;    -   DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl        ether-2′-O-bis(2-acetoxyethoxy)methyl    -   FPMP=5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl].

All of the aforementioned RNA synthesis strategies are amenable to thepresent invention. Strategies that would be a hybrid of the above e.g.using a 5′-protecting group from one strategy with a 2′-O-protectingfrom another strategy is also amenable to the present invention.

The preparation of ribonucleotides and oligomeric compounds having atleast one ribonucleoside incorporated and all the possibleconfigurations falling in between these two extremes are encompassed bythe present invention. The corresponding oligomeric comounds can behybridized to further oligomeric compounds includingoligoribonucleotides having regions of complementarity to formdouble-stranded (duplexed) oligomeric compounds. Such double strandedoligonucleotide moieties have been shown in the art to modulate targetexpression and regulate translation as well as RNA processsing via anantisense mechanism. Moreover, the double-stranded moieties may besubject to chemical modifications (Fire et al., Nature, 1998, 391,806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene,2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431;Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507;Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). Forexample, such double-stranded moieties have been shown to inhibit thetarget by the classical hybridization of antisense strand of the duplexto the target, thereby triggering enzymatic degradation of the target(Tijsterman et al., Science, 2002, 295, 694-697).

The methods of preparing oligomeric compounds of the present inventioncan also be applied in the areas of drug discovery and targetvalidation. The present invention comprehends the use of the oligomericcompounds and preferred targets identified herein in drug discoveryefforts to elucidate relationships that exist between proteins and adisease state, phenotype, or condition. These methods include detectingor modulating a target peptide comprising contacting a sample, tissue,cell, or organism with the oligomeric compounds of the presentinvention, measuring the nucleic acid or protein level of the targetand/or a related phenotypic or chemical endpoint at some time aftertreatment, and optionally comparing the measured value to a non-treatedsample or sample treated with a further oligomeric compound of theinvention. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa particular disease, condition, or phenotype.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature (2001), 411,494-498; Nishikura et al., Cell (2001), 107, 415-416; and Bass et al.,Cell (2000), 101, 235-238.)

TARGETS OF THE INVENTION

“Targeting” an antisense oligomeric compound to a particular nucleicacid molecule, in the context of this invention, can be a multistepprocess. The process usually begins with the identification of a targetnucleic acid whose function is to be modulated. This target nucleic acidmay be, for example, a cellular gene (or mRNA transcribed from the gene)whose expression is associated with a particular disorder or diseasestate, or a nucleic acid molecule from an infectious agent.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid. The terms region,segment, and site can also be used to describe an oligomeric compound ofthe invention such as for example a gapped oligomeric compound having 3separate segments.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding a nucleic acid target, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense oligomeric compounds of thepresent invention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense oligomeric compounds targeted to,for example, DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequences.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense oligomeric compounds hybridize are hereinbelow referred to as“preferred target segments.” As used herein the term “preferred targetsegment” is defined as at least an 8-nucleobase portion of a targetregion to which an active antisense oligomeric compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target segments represent accessible portions of the targetnucleic acid for hybridization.

The antisense oligomeric compounds of the present invention includeoligomeric compounds that comprise at least the 8 consecutivenucleobases from the 5′-terminus of a targeted nucleic acid e.g. acellular gene or mRNA transcribed from the gene (the remainingnucleobases being a consecutive stretch of the same oligonucleotidebeginning immediately upstream of the 5′-terminus of the antisensecompound which is specifically hybridizable to the target nucleic acidand continuing until the oligonucleotide contains from about 8 to about80 nucleobases). Similarly preferred antisense oligomeric compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains from about 8 to about 80nucleobases). One having skill in the art armed with the preferredantisense compounds illustrated herein will be able, without undueexperimentation, to identify further preferred antisense compounds.

Once one or more target regions, segments or sites have been identified,antisense oligomeric compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

In accordance with one embodiment of the present invention, a series ofpreferred compositions of nucleic acid duplexes comprising the antisenseoligomeric compounds of the present invention and their complements canbe designed for a specific target or targets. The ends of the strandsmay be modified by the addition of one or more natural or modifiednucleobases to form an overhang. The sense strand of the duplex is thendesigned and synthesized as the complement of the antisense strand andmay also contain modifications or additions to either terminus. Forexample, in one embodiment, both strands of the duplex would becomplementary over the central nucleobases, each having overhangs at oneor both termini.

In one embodiment, a duplex comprising an antisense oligomeric compoundhaving the sequence CGAGAGGCGGACGGGACCG wherein the complement strandcomprises two or more separate strands having a two-nucleobase overhangof deoxythymidine(dT) on the 3′-terminus of the antisense strand and thesame overhang on the section of the complement (sense) strand would havethe following structure:     cgagaggcggacgggaccgdTdT Antisense Strand    ||||||||||||||||||| dTdTgctctccgcctgccctggc Complement Strand

A further embodiment includes compounds having the following structurewhere there are more than one small complement strands of equal orvarying sizes from 2 to 7 nucleobases in length. cgagaggcggacgggaccgAntisense Strand |||  |||   |||  ||| gct  ccg   gcc  ggc ComplementStrands

A further embodiment includes compounds having the following structurewhere there are two complement strands of equal or varying sizes butunlinked at one point. cgagaggcggacgggaccg Antisense Strand||||||||| ||||||||| gctctccgc tgccctggc Complement Strand

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from various RNA synthesis companies such as for exampleDharmacon Research Inc., (Lafayette, Colo.). Once synthesized, thecomplementary strands are annealed. The single strands are aliquoted anddiluted to a concentration of 50 uM. Once diluted, 30 uL of each strandis combined with 15 uL of a 5× solution of annealing buffer. The finalconcentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. Thissolution is incubated for 1 minute at 90° C. and then centrifuged for 15seconds. The tube is allowed to sit for 1 hour at 37° C. at which timethe dsRNA duplexes are used in experimentation. The final concentrationof the dsRNA compound is 20 uM. This solution can be stored frozen (−20°C.) and freeze-thawed up to 5 times.

Once prepared, the desired synthetic duplexs are evaluated for theirability to modulate target expression. When cells reach 80% confluency,they are treated with synthetic duplexs comprising at least oneoligomeric compound of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired dsRNA compound at a finalconcentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional oligomeric compoundsthat modulate the expression of a target. “Modulators” are thoseoligomeric compounds that decrease or increase the expression of anucleic acid molecule encoding a target and which comprise at least an8-nucleobase portion which is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encoding a targetwith one or more candidate modulators, and selecting for one or morecandidate modulators which decrease or increase the expression of anucleic acid molecule encoding a target. Once it is shown that thecandidate modulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a nucleic acid moleculeencoding a target, the modulator may then be employed in furtherinvestigative studies of the function of a target, or for use as aresearch, diagnostic, or therapeutic agent in accordance with thepresent invention.

The preferred target segments of the present invention may also becombined with their respective complementary antisense oligomericcompounds of the present invention to form stabilized double-stranded(duplexed) oligonucleotides.

Hybridization

In the context of this invention, “hybridization” occurs when twosequences come together with enough base complementarity to form adouble stranded region. The source of the two sequences can be syntheticor native and can occur in a single strand when the strand has regionsof self complementarity. In the present invention, the preferredmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds or between an oligomeric compound and atarget nucleic acid. For example, adenine and thymine are complementarynucleobases which pair through the formation of hydrogen bonds.Hybridization can occur under varying circumstances.

An antisense oligomeric compound is specifically hybridizable whenbinding of the compound to the target nucleic acid interferes with thenormal function of the target nucleic acid to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense oligomeric compound to non-targetnucleic acid sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and under conditions in which assaysare performed in the case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which an oligomericcompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will vary with different circumstances and in thecontext of this invention, “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing of two nucleobases regardless of where the two are located. Forexample, if a nucleobase at a certain position of an oligomeric compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, the target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleobases which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleobases such that stable and specificbinding occurs between the oligonucleotide and a target nucleic acid.

It is understood in the art that the sequence of an antisense oligomericcompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. Moreover, an oligonucleotide mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). It is preferred that the antisenseoligomeric compounds of the present invention comprise at least 70%sequence complementarity to a target region within the target nucleicacid, more preferably that they comprise 90% sequence complementarityand even more preferably comprise 95% sequence complementarity to thetarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense oligomeric compound in which 18 of20 nucleobases of the antisense oligomeric compound are complementary toa target region, and would therefore specifically hybridize, wouldrepresent 90 percent complementarity. In this example, the remainingnoncomplementary nucleobases may be clustered or interspersed withcomplementary nucleobases and need not be contiguous to each other or tocomplementary nucleobases. As such, an antisense oligomeric compoundwhich is 18 nucleobases in length having 4 (four) noncomplementarynucleobases which are flanked by two regions of complete complementaritywith the target nucleic acid would have 77.8% overall complementaritywith the target nucleic acid and would thus fall within the scope of thepresent invention. Percent complementarity of an antisense oligomericcompound with a region of a target nucleic acid can be determinedroutinely using BLAST programs (basic local alignment search tools) andPowerBLAST programs known in the art (Altschul et al., J. Mol. Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

Screening and Target Validation

In a further embodiment, “preferred target segments” may be employed ina screen for additional oligomeric compounds that modulate theexpression of a selected protein. “Modulators” are those oligomericcompounds that decrease or increase the expression of a nucleic acidmolecule encoding a protein and which comprise at least an 8-nucleobaseportion which is complementary to a preferred target segment. Thescreening method comprises the steps of contacting a preferred targetsegment of a nucleic acid molecule encoding a protein with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich decrease or increase the expression of a nucleic acid moleculeencoding a protein. Once it is shown that the candidate modulator ormodulators are capable of modulating (e.g. either decreasing orincreasing) the expression of a nucleic acid molecule encoding apeptide, the modulator may then be employed in further investigativestudies of the function of the peptide, or for use as a research,diagnostic, or therapeutic agent in accordance with the presentinvention.

The preferred target segments of the present invention may also becombined with their respective complementary antisense oligomericcompounds of the present invention to form stabilized double-stranded(duplexed) oligonucleotides. Such double stranded oligonucleotidemoieties have been shown in the art to modulate target expression andregulate translation as well as RNA processsing via an antisensemechanism. Moreover, the double-stranded moieties may be subject tochemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmonsand Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al.,Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., GenesDev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498;Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target (Tijsterman etal., Science, 2002, 295, 694-697).

The compositions comprising oligomeric compounds of the presentinvention can also be applied in the areas of drug discovery and targetvalidation. The present invention comprehends the use of the oligomericcompounds and preferred targets identified herein in drug discoveryefforts to elucidate relationships that exist between proteins and adisease state, phenotype, or condition. These methods include detectingor modulating a target peptide comprising contacting a sample, tissue,cell, or organism with the oligomeric compounds of the presentinvention, measuring the nucleic acid or protein level of the targetand/or a related phenotypic or chemical endpoint at some time aftertreatment, and optionally comparing the measured value to a non-treatedsample or sample treated with a further oligomeric compound of theinvention. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa particular disease, condition, or phenotype.

Effect of nucleoside modifications on RNAi activity is evaluatedaccording to existing literature (Elbashir et al., Nature (2001), 411,494-498; Nishikura et al., Cell (2001), 107, 415-416; and Bass et al.,Cell (2000), 101, 235-238.)

Kits, Research Reagents, Diagnostics, and Therapeutics

The compositions of oligomeric compounds of the present invention can beutilized for diagnostics, therapeutics, prophylaxis and as researchreagents and kits. Furthermore, antisense oligonucleotides, which areable to inhibit gene expression with exquisite specificity, are oftenused by those of ordinary skill to elucidate the function of particulargenes or to distinguish between functions of various members of abiological pathway.

For use in kits and diagnostics, the compositions of the presentinvention, either alone or in combination with other oligomericcompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense oligomeric compounds are compared tocontrol cells or tissues not treated with antisense oligomeric compoundsand the patterns produced are analyzed for differential levels of geneexpression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds and or oligomeric compounds that affectexpression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compositions of the invention are useful for research anddiagnostics in one sense because the oligomeric compounds of thecompositions hybridize to nucleic acids encoding proteins. For example,oligonucleotides that are shown to hybridize with such efficiency andunder such conditions as disclosed herein as to be effective proteininhibitors will also be effective primers or probes under conditionsfavoring gene amplification or detection, respectively. These primersand probes are useful in methods requiring the specific detection ofnucleic acid molecules encoding proteins and in the amplification of thenucleic acid molecules for detection or for use in further studies.Hybridization of the antisense oligonucleotides, particularly theprimers and probes, of the invention with a nucleic acid can be detectedby means known in the art. Such means may include conjugation of anenzyme to the oligonucleotide, radiolabelling of the oligonucleotide orany other suitable detection means. Kits using such detection means fordetecting the level of selected proteins in a sample may also beprepared.

The specificity and sensitivity of antisense methodologies is alsoharnessed by those of skill in the art for therapeutic uses. Antisenseoligomeric compounds have been employed as therapeutic moieties in thetreatment of disease states in animals, including humans. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that antisense oligomericcompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofa selected protein is treated by administering compositions of theinvention in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a protein inhibitor. The protein inhibitors of the present inventioneffectively inhibit the activity of the protein or inhibit theexpression of the protein. In one embodiment, the activity or expressionof a protein in an animal is inhibited by about 10%. Preferably, theactivity or expression of a protein in an animal is inhibited by about30%. More preferably, the activity or expression of a protein in ananimal is inhibited by 50% or more. For example, the reduction of theexpression of a protein may be measured in serum, adipose tissue, liveror any other body fluid, tissue or organ of the animal. Preferably, thecells contained within the fluids, tissues or organs being analyzedcontain a nucleic acid molecule encoding a protein and/or the proteinitself

The compositions of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount to a suitablepharmaceutically acceptable diluent or carrier. Use of the compositionsand methods of the invention may also be useful prophylactically.

Formulations

The compositions of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The compositions of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of thecompositions of the invention, pharmaceutically acceptable salts of suchprodrugs, and other bioequivalents. The term “prodrug” indicates atherapeutic agent that is prepared in an inactive form that is convertedto an active form (i.e., drug) within the body or cells thereof by theaction of endogenous enzymes or other chemicals and/or conditions. Inparticular, prodrug versions of the oligonucleotides of the inventionare prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivativesaccording to the methods disclosed in WO 93/24510 to Gosselin et al.,published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 toImbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the oligomeric compounds of theinvention: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations which include the compositions of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. applications Ser. No. 09/108,673 (filed Jul. 1, 1998),Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filedFeb. 8, 2002, each of which is incorporated herein by reference in theirentirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more of the compositions of the invention and one ormore other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to cancer chemotherapeutic drugs such as daunorubicin,daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine ara-binoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethyl-melamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclo-hexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compositions of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of compositions of the invention and other non-antisensedrugs are also within the scope of this invention. One or morecompositions of the invention can be used in combination with othertherapeutic agents to create a coctail as is currently the strategy forcertain viral infections.

In another related embodiment, therapeutically effective combinationtherapies may comprise the use of two or more compositions of theinvention wherein the multiple compositions are targeted to a single ormultiple nucleic acid targets. Numerous examples of antisense oligomericcompounds are known in the art. Two or more combined compounds may beused together or sequentially

Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLE 1

Duplex Constructs having Short Sense Strands Bound to 20 mer AntisenseStrands

The activity of different double strand constructs having short sensestrands compared to the antisense strands was determined. The doublestrand constructs comprised 20 mer antisense strands having one or two10 mer sense strands hybridized thereto. The study examined three basicmotifs having the short sense strand hybridized at the 3′-end, the5′-end or having 2 short sense strands hybridized one at the 3′-end andone at the 5′-end. The activity of the unmodified-antisense strand wascompared to various chemically modified antisense strands using thethree basic motifs. The study compared these double strand constructs tountreated and positive controls for their ability to inhibit PTEN mRNAlevels in HUVEC cells. ISIS# SEQ ID No: Sequence 5′-3′ 308746 1AAGUAAGGACCAGAGACAAA (complete sense) 334471 2 AAGUAAGGAC (short sense)334472 3 CAGAGACAAA (short sense) 303912 45′-P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*U*A*C*U*U (as) 317502 45′-P-2′-F-U*2′-F-U*2′-F-U*G*2′-F-C*2′-F-U*C*U*G*G*U*C*2′F-C*2′F-U*2′-F-U*A*2′-F-C*2′-F-U*U (as) 319022 45′-P-2′-F-U*2′-F-U*2′-F-U*2′-F-G*2′-F-U*2′-F-C*2′-F-U*2′-F-C*2′-F-U*2′-F-G*2′-F-G*2′-F-U*2′-F-C*2′-F-C*2′-F-U*2′-F-U*2′-F-A*2′-F-C*2′-F-U*2′-F-U(as)

Constructs (AS/S) Activity (1, 5, 25 and 75 nM) 303912/none 115.5 107.345.5 31.7 303912/308746 18.5 15.0 26.8 30.2 303912/334471 and 33447258.8 41.7 34.7 36.3 303912/334471 115.5 97.6 25.6 38.7 303912/334472136.0 80.4 31.6 38.7 317502/none 67.6 82.4 53.8 63.7 317502/308746 13.414.6 53.1 54.6 317502/334471 and 334472 27.2 16.1 73.2 77.6317502/334471 124.9 83.3 72.8 69.3 317502/334472 72.6 41.8 46.9 54.0319022/none 104.2 140.9 126.0 115.5 319022/308746 60.4 73.4 118.5 118.2319022/334471 and 334472 57.6 33.2 58.9 57.8 319022/334471 127.0 124.4140.4 132.9 319022/334472 63.3 59.3 78.3 95.5none phosphodiester*Phosphorothioate2′-F 2′-Fluorine

Constructs S 5′-3′, AS 3′-5′ 334471 short sense strandXXXXXXXXXXXXXXXXXXXX antisense strand 334472 short sense strandXXXXXXXXXXXXXXXXXXXX antisense strand 334471 334472 2 sense strandsXXXXXXXXXXXXXXXXXXXX antisense strand.

EXAMPLE 2

Constructs having Antisense Strands with Linked Sense Strands BoundThereto

The activity of different double strand constructs having short sensestrands linked to a region of an antisense strand was determined. Thedouble strand constructs comprise a 20 mer antisense strand having oneor two 10 mer sense strands hybridized thereto. The 10 mer sense strandsare covalently linked to the antisense strand through a linking moietyand are hybridized to a region of the antisene strand thereby forming aduplex region along the antisense strand. The study examined three basicmotifs having the covalently linked short sense strands hybridized atthe 3′ end, the 5′ end or having 2 covalently linked short sense strandshybridized with one at the 3′ end and the other at the 5′ end. Theactivity of the unmodified antisense strand was compared to variouschemically modified antisense strands using the three basic motifs. Thestudy compared these double strand constructs to untreated and positivecontrols for their ability to inhibit PTEN mRNA levels in HUVEC cells.ISIS# SEQ ID No: Sequence 5′-3′ 271784 5 GUAAGGACCAGAGACAAAdTdT (s)297803 6 UUUGUCUCUGGUCCUUACUdTdT (as) 317469 4CAGAGACAAA-18s-UUUGUCUCUGGUCCUUACUU-18s-AAGUAAGGAC (s-18s-as-18s-s)317470 4 CAGAGACAAA-18s-UUUGUCUCUGGUCCUUACUU- (s-18s-as, 5′-hairpin)317471 4 UUUGUCUCUGGUCCUUACUU-18s-AAGUAAGGAC (as-18s-s, 3′-hairpin)none -phosphodiester

-   -   *—Phosphorothioate    -   F—Fluorine

18s—18 atom peg spacer Constructs (AS/S) % Activity (50 nM) vs untreatedcontrol 297803/271784 12 317469 72 317470 75 317471 107.

EXAMPLE 3

Duplex Constructs having Short Sense Strands Bound to Antisense Strands

The activity of different double strand constructs having short sensestrands compared to the antisense strands was determined. The doublestrand constructs comprised 20 mer antisense strands having one shortstrand hybridized at the 3′-end or one short strand hybridized at the5′-end of the antisense oligomeric compound. The study examined thesetwo basic motifs for a wide variety of antisense oligomeric compounds.The activity of the composition having the unmodified antisense strandwas compared to various chemically modified antisense strands using thethree basic motifs. The study compared these constructs to untreated andpositive controls for their ability to inhibit PTEN mRNA levels in HUVECcells. ISIS# SEQ ID No: (activities) Sequence 5′-3′ (AS) 335449 4 (7.6,19.2) P-U.U.U.G.U.C.U.C.U.G.G.U.C.C.U.U.A.C.U.U 303912 4 (10.1, 23.0)P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*U*A* C*U*U 317502 4 (66.8, 43.5)P-2′-F-U*2′-F-U*2′-F-U*G*2′-F-U*2′-F-C*2′-F-U*C*U*G*G*U*C*2′-F-C*2′-F-U*2′-F-U*A*2′-F-C*2′F- U*U 319022 4 (58.3,44.8) P-2′-F-U*2′-F-U*2′-F-U*2′-F-G*2′-F-U*2′-F-C*2′-F-U*2′-F-C*2′-F-U*2′-F-G*2′-F-G*2′-F-U*2′-F-C*2′-F-C*2′-F-U*2′-F-U*2′-F-A*2′-F-C*2′-F-U*2′-F-U 330916 7 (88.0, 54.7)P-U*U*U*G*U*C*U*C*U*G*G*(MOE)T*(MOE)C [5Me]*(MOE)C[5Me]*U*U*A*C*U*U335226 4 (11.2, 59.8) P-U*U*U*G*U*mC*mU*mC*U*G*G*U*C*C*U* U*A*C*U*U331426 4 (57.5, 60.1) P-U*U*U*G*U*C*U*C*(LNA)U*(LNA)G*(LNA)G*U*C*C*U*U*A*C*U*U 331427 4 (76.8, 60.1)P-U*U*U*G*U*(LNA)C*(LNA)U*(LNA)C*U*G*G* U*C*C*U*U*A*C*U*U 340570 4(67.6, 60.2) P-mU*2′-F-U*mU*2′-F-G*mU*2′-F-C*mU*2′-F-C*mU*2′-F-G*mG*2′-F-U*mC*2′-F-C*mU*2′-F- U*mA*2′-F-C*mU*2′-F-U 317468 4(114.8, 61.8) P-2′-F-U.2′-F-U.2′-F-U.G.U.C.U.C.U.G.G.U.C.C.U.U.A.2′-F-C.2′-F-U.U 334254 4 (60.1, 63.1)P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*2′-F-U*2′ F-A*2′-F-C*2′-F-U*2′-F-U317466 4 (94.7, 63.3) P-2′-F-U*2′-F-U*2′-F-U*G*U*C*U*C*U*G*G*U*C*C*U*U*A*2′-F-C*2′-F-U*U 333756 4 (71.9, 65.8)P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*U*A*2′-F- C*2′-F-U*2′-F-U 319013 8(84.2, 68.9) P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*mU* U*mU 330915 9 (78.2,69.2) P-*U*U*G*U*C*U*C*U*G*G*U*C*C*(MOE)T* *(MOE)A*C*U*U 331428 4 (81.4,69.5) P-U*U*(LNA)U*(LNA)G*(LNA)U*C*U*C*U*G*G*U* C*C*U*U*A*C*U*U 333752 4(95.2, 69.8) P-U*U*U*G*U*2′F-C*2′-F-U*2′-F-C*U*G*G* U*C*C*U*U*A*C*U*U336678 4 (113.2, 70.9) P-4′-S-U.4′-S-U.4′-S-U.G.U.C.U.C.U.G.G.U.C.C.U.U.A.C.U.U 340572 4 (75.1, 72.0)P-2′-F-U*mU*2′-F-U*mG*2′F-U*mC*2′-F-U*mC*2′-F-U*mG*2′-F-G*mU*2′-F-C*mC*2′-F-U*mU*2′F- A*mC*2′-F-U*mU 334255 4 (108.1,72.7) P-U*U*2′-F-U*2′-F-G*2′-F-U*C*U*C*U*G*G*U* C*C*U*U*A*C*U*U 319018 4(95.2, 72.7) P-2′-F-U.2′-F-U.2′-F-U.2′-F-G.2′-F-U.2′-F-C.2′-F-U.2′-F-C.2′-F-U.2′-F-G.2′-F-G.2′-F-U.2′-F-C.2′-F-C.2′-F-U.2′-F-U.2′-F-A.2′-F-C.2′-F-U.2′-F-U 330918 10 (65.8, 73.0)P-U*U*U*G*U*(MOE)C[5Me]*(MOE)T*(MOE)C [5Me]*U*G*G*U*C*C*U*U*A*C*U*U319017 11 (85.1, 73.0) P-U*U*U*G*U*C*U*C*U*G*dG*dT*dC*dC*dT*dT*dA*mC*mU*mU 331695 4 (69.6, 73.1) P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*U*A*(LNA)C*(LNA)U*(LNA)U 316449 4 (84.5, 73.8)P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U* U*A*mC*mU*mU 329392 12 (117.5, 74.2)P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*U*A* (MOE)C[5Me]*(MOE)T*(MOE)T 340653 4(68.0, 75.6) P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*(LNA)U* (LNA)U*(LNA)A*C*U*U334253 4 (78.2, 75.9) P-U*U*U*G*U*C*U*C*2′-F-U*2′-F-G*2′-F-G*U*C*C*U*U*A*C*U*U 335216 13 (64.8, 76.5)P-(MOE)T*dU*(MOE)T*dG*(MOE)T*dC[5Me]*(MOE)T*dC[5Me]*(MOE)T*dG*(MOE)G*dU*(MOE)C[5Me]*dC[5Me]*(MOE)T*dU*(MOE)A* dC[5Me]*(MOE)T*dU 319015 4 (93.5,77.2) P-U*U*U*G*U*C*U*C*U*G*G*mU*mC*mC*mU* mU*mA*mC*mU*mU 333749 4(73.3, 77.6) P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*2′-F-U*2′-F- U*2′-F-A*C*U*U303912 4 (96.5, 79.4) P-U*U*U*G*U*C*U*C*U*G*G*U*C*C* U*U*A*C*U*U 3352244 (85.7, 79.6) P-U*U*U*G*U*C*U*C*U*G*G*mU*mC* mC*U*U*A*C*U*U 336238 4(68.6, 79.9) P-U*U*U*G*U*C[5Me]*U*C[5Me]*U*G*G*U*C[5Me]*C[5Me]*U*U*A*C[5Me]*U*U 342852 4 (91.3, 80.7)P-4′-S-U.4′-S-U.4′-S-U.G.4′-S-U.C.U.C.U.G.G.U.C.C.U.4′-S-U.A.4′-S-C.4′-S-U.4′-S-U 334257 4 (81.1, 81.0)P-2′-F-U*2′-F-U*2′-F-U*G*U*C*U*C*U*G*G* U*C*C*U*U*A*C*U*U 336239 4(61.2, 81.3) P-U*U*U[5Br]*G*U*C*U*C*U*G*G*U*C* C*U[5Br]*U*A*C*U*U 3362404 (77.5, 81.4) P-U*U*U*G*U*C*U*2′-F-C*2′-F-U*G*G*2′-F-U*2′-F-C*C*U*U*A*mC*mU*mU 335225 4 (101.8, 81.7)P-U*U*U*G*U*C*U*C*mU*mG*mG*U*C* C*U*U*A*C*U*U 303913 14 (134.7, 82.0)mG*mU*mC*mU*C*U*G*G*U*C*C*U* U*mA*mC*mU*mU 316449 4 (81.9, 82.3)P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*U*A*mC* mU*mU 331430 4 (105.0, 82.4)P-(LNA)U*(LNA)U*(LNA)U*G*U*C*U*C* U*G*G*U*C*C*U*U*A*C*U*U 319016 15(91.2, 84.8) P-U*dT*dT*dG*dT*dC*dT*dC*U*G*G* U*C*C*U*U*A*mC*mU*mU 3405694 (60.2, 85.0) P-mU.2′-F-U.mU.2′-F-G.mU.2′-F-C.mU.2′-F-C.mU.2′-F-G.mG.2′-F-U.mC.2′-F-C.mU.2′-F-U.mA.2′-F-C.mU.2t-F-U 336676 4 (106.1,85.4) P-U.U.U.G.U.C.4′-S-U.4′-S-C.4′-S-U.G.G.U. C.C.U.U.A.C.U.U 331694 4(87.6, 86.0) P-U*U*U*G*U*C*U*C*U*G*G*(LNA)U*(LNA)C* (LNA)C*U*U*A*C*U*U333750 4 (86.8, 87.5) P-U*U*U*G*U*C*U*C*U*G*G*2′-F-U*2′-F-C*2′-F-C*U*U*A*C*U*U 335117 16 (90.8, 87.6)P-2′-F-U*2′-F-U*2′-F-U*2′-F-G*2′-F-U*2′-F-C*2′-F-U*2′-F-C*2′-F-U*2′-F-C*2′-F-C*2′-F-U*2′-F-U*2′-F-A*2′-F- C*2′-F-U*2′-F-U319014 4 (98.2, 87.8) P-U*U*U*G*U*C*U*C*U*G*G*U*C*mC* mU*mU*mA*mC*mU*mU335215 17 (83.7, 88.0) P-dU*(MOE)T*dU*(MOE)G*dU*(MOE)C[5Me]*dU*(MOE)C[5Me]*dU*(MOE)G*dG*(MOE)T*dC[5Me]* (MOE)C[5Me]*dU*(MOE)T*dA*(MOE)C[5Me]*dU*(MOE)T 339923 18 (67.4, 88.2)P-dT.2′-F-U.dT.2′-F-G.dT.2′-F-C.dT.2′-F-C.dT.2′-F-2′-F-U.dC[5Me]2′-F-C.dT.2′-F-U.dA.2′-F-C.dT.2′-F-U 335217 4 (81.5, 88.5)P-dU*mU*dU*mG*dU*mC*dU*mC*dU*mG*dG* mU*dC*mC*dU*mU*dA*mC*dU*mU 330917 19(102.2, 88.5) P-U*U*U*G*U*C*U*C*(MOE)T*(MOE)G* (MOE)G*U*C*C*U*U*A*C*U*U335227 4 (167.1, 88.7) P-U*U*mU*mG*mU*C*U*C*U*G*G*U*C* C*U*U*A*C*U*U335456 4 (83.3, 88.8) P-mU.U.mU.G.mU.C.mU.C.mU.G.mG.U.mC.C.mU.U.mA.C.mU.U 335220 17 (96.5, 88.8)P-dU.(MOE)T.dU.(MOE)G.dU.(MOE)C[5Me].dU.(MOE)C[5Me].dU.(MOE)G.dG.(MOE)T.dC [5Me].(MOE)C[5Me].dU.(MOE)T.dA.(MOE)C[5Me].dU.(MOE)T 335454 4 (79.2, 89.2)P-mU*U*mU*G*mU*C*mU*C*mU*G*mG*U* mC*C*mU*U*mA*C*mU*U 336674 4 (124.8,90.0) P-U.U.U.G.U.C.U.C.U.G.G.U.C.C.U.4′-S-U.A.4′-S-C.4′-S- U.4′-S-U335201 20 (123.2, 92.7) P-dT.(MOE)T.dT.(MOE)G.dT.(MOE)C[5Me].dT.(MOE)C[SMe].dT.(MOE)G.dG.(MOE)T.dC [5Me].(MOE)C[5Me].dT.(MOE)T.dA.(MOE)C[5Me].dT.(MOE)T 328795 4 (66.6, 92.9) P-U*U*U*mG*mU*mC*mU*C*U*G*G*U*C*C*U*U*mA*mC*mU*mU 329391 12 (121.1, 93.5)P-U*U*U*G*U*C*U*C*U*G*G*U*C* C*U*(MOE)U*(MOE)A*(MOE)C[5Me]*(MOE)T*(MOE)T 335449 4 (124.1, 93.7)P-U.U.U.G.U.C.U.C.U.G.G.U.C.C.U.U.A.C.U.U 340571 4 (118.9, 94.2)P-2′-F-U.mU.2′-F-U.mG.2′-F-U.mC.2′-F-U.mC.2′-F-U.mG.2′-F-G.mU.2′-F-C.mC.2′-F-U.mU.2′-F-A.mC.2′-F- U.mU 339924 13 (68.4,95.5) P-dT*2′-F-U*dT*2′-F-G*dT*2′-F-C*dT*2′-F-C*dT*2′-F-G*dG*2′-F-U*dC[5Me]*2′-F-C*dT*2′-F-U*dA*2′-F- C*dT*2′-F-U 335197 20(112.4, 96.0) P-dT*(MOE)T*dT*(MOE)G*dT*(MOE)C[5Me]*dT*(MOE)C[5Me]*dT*(MOE)G*dG*(MOE)T *dC[5Me]*(MOE)C[5Me]*dT*(MOE)T*dA*(MOE)C[5Me]*dT*(MOE)T 334469 4 (112.9, 96.8)mU.mU.mU.mG.mU.mC.mU.mC.mU.mG.mG.mU.m C.mC.mU.mU.mA.mC.mU.mU 336671 4(118.5, 97.5) P-U.U.U.G.U.C.U.C.U.G.G.U.C.C.U.U.A.4′-S-C.4′-S- U.4′-S-U336672 4 (111.5, 98.3) P-U.U.U.G.U.C.U.C.U.G.G.U.4′-S-C.4′-S-C.4′-S-U.U.A.C.U.U 335209 20 (82.3, 98.9) P*(MOE)T*(MOE)T*(MOE)T*dG*dT*(MOE)C[5Me]*dT*dC[5Me]*(MOE)T*dG*dG*(MOE)T*dC[5Me]*dC[5Me]*(MOE)T*dT*dA*(MOE)C [5Me]*(MOE)T*(MOE)T 330919 21 (71.6,100.2) P-U*U*(MOE)T*(MOE)G*(MOE)T*C*U*C*U* G*G*U*C*C*U*U*A*C*U*U 3352234 (129.8, 100.8) P-U*U*U*G*U*C*U*C*U*G*G*U* C*C*mU*mU*mA*C*U*U 303912 4(102.5, 100.8) P-U*U*U*G*U*C*U*C*U*G*G*U* C*C*U*U*A*C*U*U 335228 4(119.9, 101.4) P-mU*mU*mU*G*U*C*U*C*U*G*G*U* C*C*U*U*A*C*U*U 335198 20(104.2, 103.1) P-(MOE)T*dT*(MOE)T*dG*(MOE)T*dC[5Me]*(MOE)T*dC[5Me]*(MOE)T*dG*(MOE)G*dT*(MOE)C[5Me]*dC[5Me]*(MOE)T*dT*(MOE)A* dC[5Me]*(MOE)T*dT 335221 4 (140.8,103.6) P-dU.mU.dU.mG.dU.mC.dU.mC.dU.mG.dG.m U.dC.mC.dU.mU.dA.mC.dU.mU336675 4 (155.7, 103.9) P-U.U.U.G.U.C.U.C.U.G.G.U.C.C.U.U.A.C.U.4′-S-U330997 22 (114.1, 107.0) P-(MOE)T*(MOE)T*(MOE)T*G*U*C*U*C*U*G*G*U*C*C*U*U*A*C*U*U 339925 17 (135.9, 107.0)P-2′-F-U.dT.2′-F-U.dG.2′-F-U.dC[5Me].2′-F-U.dC[5Me].2′-F-U.dG.2′-F-G.dT.2′-F-C.dC[5Me].2′-F-U.dT.2′-F-A.dC[5Me].2′-F-U.dT 328794 4 (85.5, 108.7)P-mU*mU*U*G*U*C*U*C*U*G* G*U*C*C*U*U*A*C*U*U 339926 17 (92.6, 109.5)P-2′-F-U.dT.2′-F-U.dG.2′-F-U.dC[5Me].2′-F-U*dC[5Me]*.2′-F-U*dG*2′-F-G*dT*2′-F-C*dC[5Me]*2′-F-U*dT*2′-F-A*dC[5Me]*2′-F-U*dT 308743 4 (116.4, 110.0)P-mU*mU*mU*G*U*C*U*C*U*G* G*U*C*C*U*U*A*mC*mU*mU 335210 20 (102.4,110.6) P-(MOE)T.(MOE)T.(MOE)T.dG.dT.(MOE)C[5Me].dT.dC[5Me].(MOE)T.dG.dG.(MOE)T.dC[5Me].dC[5Me].(MOE)T.dT.dA.(MOE)C [5Me].(MOE)T.(MOE)T 334464 4 (130.7,110.6) 18S.U.U.U.G.U.C.U.C.U.G.G.U.C.C.U.U.A.C.U.U 335457 4 (125.5,111.9) P-U.mU.U.mG.U.mC.U.mC.U.mG.G.m U.C.mC.U.mU.A.mC.U.mU 336673 4(118.2, 115.3) P-U.U.U.G.U.C.U.C.U.G.G.4′-S-U.4′-S-C.4′-S- C.U.U.A.C.U.U335202 20 (152.9, 115.6) P-(MOE)T.dT.(MOE)T.dG.(MOE)T. dC[5Me].(MOE)T.dC[5Me].(MOE)T.dG.(MOE)G.dT.(MOE)C]5Me].dC[5Me].(MOE)T.dT.(MOE)A. dC[5Me].(MOE)T.dT 335218 4 (91.7,117.7) P-mU*dU*mU*dG*mU*dC*mU*dC*mU*dG* mG*dU*mC*dC*mU*dU*mA*dC*mU*dU342851 4 (117.1, 118.1) P-4′-S-U.4′-S-U.4′-S-U.G.4′-S-U.C.U.C.U.G.G.U.C.C.U.4′-S-U.A.4′-S-C.4′-S-U.4′-S-U 335455 4 (140.4, 121.3)P-U*mU*U*mG*U*mC*U*mC*U*mG* G*mU*C*mC*U*mU*A*mC*U*mU 335222 4 (143.4,129.6) P-mU.dU.mU.dG.mU.dC.mU.dC.mU.dG.mG.dU.m C.dC.mU.dU.mA.dC.mU.dULegend sugars 2′-OH unless otherwise marked m 2′-O-methyl F 2′-F (MOE)2′-O-(CH₂)₃-O-CH₃ (LNA) locked nucleic acid 2′-O-CH₂-4′(bicyclic sugarnucleoside) 4′-S 4′-thionucleoside C[5Me] 5-methyl C U[5Br] 5-BromoUperiod phosphodiester asterick phosphorothioate dN deoxy N (N = G, dGdeoxyguanidine) 18s 18 atom peg spacer P- terminal phosphate group

The percent activities shown in brackets under the SEQ ID NO headinglist the relative activities for the constructs shown below. Theactivity for the construct having the 334471 short sense strand islisted first followed by the activity for the construct having the334472 short sense strand. Constructs S 5′-3′, AS 3′-5′ 334471 shortsense strand XXXXXXXXXXXXXXXXXXXX antisense strand variable length334472 short sense strand XXXXXXXXXXXXXXXXXXXX antisense strand variablelength.

EXAMPLE 4

Activity of Duplex Constructs having Modified Sense and/or AntisenseStrands in Primary Mouse Hepatocytes

The activity of different double strand constructs having short sensestrands compared to the antisense strands was determined. The doublestrand constructs comprised 20 mer antisense strands having one or two10 mer sense strands hybridized thereto. The study examined three basicmotifs having the short sense strand hybridized at the 3′-end, the5′-end or having 2 short sense strands hybridized one at the 3′-end andone at the 5′-end. The activity of the unmodified antisense strand wascompared to various chemically modified antisense strands using thethree basic motifs. In addition, modified sense strands were used inplace of unmodified sense strands to determine their activities. Thestudy compared these double strand constructs to untreated and positivecontrols for their ability to inhibit PTEN mRNA levels in primary mousehepatocytes. ISIS# SEQ ID No: Sequence 5′-3′ 335449 4(as)P-UUUGUCUCUGGUCCUUACUU 303912 4(as)P-U*U*U*G*U*C*U*C*U*G*G*U*C*C*U*U*A*C*U*U 319022 4(as)P-2′-F-U*2′-F-U*2′-F-U*2′-F-G*2′-F-U*2′-F-C*2′-F-U*2′-F-C*2′F-U*2′-F-G*2′-F-G*2′-F-U*2′-F-C*2′-F-C*2′-F-U*2′-F-U*2′-F-A*2′-F-C*2′-F-U*2′-F-U 317502 4(as)P-2′-F-U*2′-F-U*2′-F-U*G*2′-F-U*2′-F-C*2′-F-U*C*U*G*G*U*C*2′-F-C*2′-F-U*2′-F-U*A*2′-F-C*2′-F-U*U 308746 1(s)AAGUAAGGACCAGAGACAAA 334471 2(s) AAGUAAGGAC 334472 3(s) CAGAGACAAA351297 3(s) 2′-F-CAGAGA-2′-F-CAAA 351298 26(s) AGGACCAGAG (controlunmodified) 351299 26(s) AGGA-2′-F-C-2′-F-CAGAG (control 2′-F modified)Legend: none = phosphodiester, * = phosphorothioate, 2′-F = 2′-fluorineand P = a 5′-phosphate group.

The percent inhibition of the PTEN mRNA in the assay was measured as apercent of the untreated control which was set at 100%. The relativeactivities are shown below. % untreated Antisense strand Sense strandcontrol (100%) 335449 None 70.5 335449 308746 34.4 335449 334471/72 63.1335449 334471 84.2 335449 334472 79.9 335449 351297 80.7 335449334471/351297 42.3 335449 351298 92.6 335449 351299 86.3 303912 None73.3 303912 308746 26.7 303912 351297 64.8 303912 334471/351297 50.4303912 351298 71.8 303912 351299 87.5 317502 None 96.2 317502 30874640.5 317502 334471/334472 40.8 317502 334471 83.5 317502 334472 59.4317502 351297 70.9 317502 334471/351297 43.9 317502 351298 82.7 317502351299 73.1 319022 None 114.4 319022 308746 58.7 319022 334471 106.8319022 334472 57.4 319022 351297 83.5 319022 351298 87.9

EXAMPLE 5

Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

EXAMPLE 6

Oligonucleotide and Oligonucleoside Synthesis

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidite oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S.Patents 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages are prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289,all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

EXAMPLE 7

RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand, 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense oligomeric compounds (RNA oligonucleotides) of the presentinvention can be synthesized by the methods herein or purchased fromDharmacon Research, Inc (Lafayette, Colo.). Once synthesized,complementary RNA antisense oligomeric compounds can then be annealed bymethods known in the art to form double stranded (duplexed) antisenseoligomeric compounds. For example, duplexes can be formed by combining30 μl of each of the complementary strands of RNA oligonucleotides (50uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate)followed by heating for 1 minute at 90° C., then 1 hour at 37° C. Theresulting duplexed antisense oligomeric compounds can be used in kits,assays, screens, or other methods to investigate the role of a targetnucleic acid.

EXAMPLE 8

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites. [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

EXAMPLE 9

Design and Screening of Duplexed Antisense Oligomeric Compounds Directedto a Selected Target

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense oligomeric compounds of the presentinvention and their complements can be designed to target a target. Theends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe dsRNA is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:  cgagaggcggacgggaccgTT antisense strand   |||||||||||||||||||TTgctctccgcctgccctggc complementary sense strand

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense oligomeric compounds are evaluatedfor their ability to modulate a target expression.

When cells reached 80% confluency, they are treated with duplexedantisense oligomeric compounds of the invention. For cells grown in96-well plates, wells are washed once with 200 μL OPTI-MEM-1reduced-serum medium (Gibco BRL) and then treated with 130 μL ofOPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desiredduplex antisense oligomeric compound at a final concentration of 200 nM.After 5 hours of treatment, the medium is replaced with fresh medium.Cells are harvested 16 hours after treatment, at which time RNA isisolated and target reduction measured by RT-PCR.

EXAMPLE 10

Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (±32 ±48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

EXAMPLE 11

Oligonucleotide Synthesis-96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

EXAMPLE 12

Oligonucleotide Analysis using 96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates were diluted fromthe master plate using single and multi-channel robotic pipettors.Plates were judged to be acceptable if at least 85% of the oligomericcompounds on the plate were at least 85% full length.

EXAMPLE 13

Cell Culture and Oligonucleotide Treatment

The effect of oligomeric compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with Oligomeric Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 23, full P═S, gaps are deoxy) which istargeted to human H-ras, or ISIS 18078, (GTGCGCGCGA-GCCCGAAATC, SEQ IDNO: 24, full P═S) which is targeted to human Jun-N-terminal kinase-2(JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethylsshown in bold 3/9/8 and 5/9/6 respectively) with a phosphorothioatebackbone. For mouse or rat cells the positive control oligonucleotide isISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 25, a 2′-O-methoxyethylgapmer (2′-O-methoxyethyls shown in bold, full P═S, 5/10/5) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

EXAMPLE 14

Analysis of Oligonucleotide Inhibition of a Target Expression

Antisense modulation of a target expression can be assayed in a varietyof ways known in the art. For example, a target mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are well known in theart. Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

EXAMPLE 15

Design of Phenotypic Assays and in vivo Studies for the use of a TargetInhibitors Phenotypic Assays

Once a target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the a target inhibitors.Hallmark genes, or those genes suspected to be associated with aspecific disease state, condition, or phenotype, are measured in bothtreated and untreated cells.

In vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study.

To account for the psychological effects of receiving treatments,volunteers are randomly given placebo or a target inhibitor.Furthermore, to prevent the doctors from being biased in treatments,they are not informed as to whether the medication they areadministering is a a target inhibitor or a placebo. Using thisrandomization approach, each volunteer has the same chance of beinggiven either the new treatment or the placebo.

Volunteers receive either the a target inhibitor or placebo for eightweek period with biological parameters associated with the indicateddisease state or condition being measured at the beginning (baselinemeasurements before any treatment), end (after the final treatment), andat regular intervals during the study period. Such measurements includethe levels of nucleic acid molecules encoding a target or a targetprotein levels in body fluids, tissues or organs compared topre-treatment levels. Other measurements include, but are not limitedto, indices of the disease state or condition being treated, bodyweight, blood pressure, serum titers of pharmacologic indicators ofdisease or toxicity as well as ADME (absorption, distribution,metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and a target inhibitor treatment. In general,the volunteers treated with placebo have little or no response totreatment, whereas the volunteers treated with the a target inhibitorshow positive trends in their disease state or condition index at theconclusion of the study.

EXAMPLE 16

RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

EXAMPLE 17

Real-Time Quantitative PCR Analysis of a Target mRNA Levels

Quantitation of a target mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5× PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM™ Taq, 5 UnitsMuLV reverse transcriptase, and 2.5× ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and are designed to hybridize to a human a target sequence, usingpublished sequence information.

EXAMPLE 18

Northern Blot Analysis of a Target mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human a target, a human a target specific primer probe set isprepared by PCR To normalize for variations in loading and transferefficiency membranes are stripped and probed for humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, PaloAlto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

EXAMPLE 19

Inhibition of Human a Target Expression by Oligomeric Compounds

In accordance with the present invention, a series of oligomericcompounds are designed to target different regions of the human targetRNA. The oligomeric compounds are analyzed for their effect on humantarget mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from three experiments. The targetregions to which these preferred sequences are complementary are hereinreferred to as “preferred target segments” and are therefore preferredfor targeting by oligomeric compounds of the present invention. Thesequences represent the reverse complement of the preferred oligomericcompounds.

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the oligomericcompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother oligomeric compounds that specifically hybridize to thesepreferred target segments and consequently inhibit the expression of atarget.

According to the present invention, oligomeric compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

EXAMPLE 20

Western Blot Analysis of Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

EXAMPLE 21

Representative Cell Lines

HuVEC Cells:

The human umbilical vein endothilial cell line HuVEC was obtained fromthe American Type Culture Collection (Manassas, Va.). HuVEC cells wereroutinely cultured in EBM (Clonetics Corporation Walkersville, Md.)supplemented with SingleQuots supplements (Clonetics Corporation,Walkersville, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence were maintained for up to 15passages. Cells were seeded into 96-well plates (Falcon-Primaria #3872)at a density of 10000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

MCF-7 Cells

The human breast carcinoma cell line MCF-7 is obtained from the AmericanType Culture Collection (Manassas, Va.). These cells contain a wild-typep53 gene. MCF-7 cells are routinely cultured in DMEM low glucose(Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetalcalf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells areroutinely passaged by trypsinization and dilution when they reach 90%confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872)at a density of 7000 cells/well for treatment with the oligomericcompounds of the invention.

HepB3 Cells

The human hepatoma cell line HepB3 (Hep3B2.1-7) is obtained from theAmerican Type Culture Collection (ATCC-ATCC Catalog # HB-8064)(Manassas, Va.). This cell line was initially derived from ahepatocellular carcinoma of an 8-yr-old black male. The cells areepithelial in morphology and are tumorigenic in nude mice. HepB3 cellsare routinely cultured in Minimum Essential Medium (MEM) with Earle'sBalanced Salt Solution, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate,0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate (ATCC #20-2003,Manassas, Va.) and with 10% heat-inactivated fetal bovine serum(Gibco/Life Technologies, Gaithersburg, Md.). Cells are routinelypassaged by trypsinization and dilution when they reach 90% confluence.

T-24 Cells

The transitional cell bladder carcinoma cell line T-24 is obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellsare routinely cultured in complete McCoy's 5A basal media (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units permL, and streptomycin 100 μg/mL (Gibco/Life Technologies, Gaithersburg,Md.). Cells are routinely passaged by trypsinization and dilution whenthey reach 90% confluence. Cells are seeded into 96-well plates(Falcon-Primaria #3872) at a density of 7000 cells/well for treatmentwith the compound of the invention.

A549 Cells

The human lung carcinoma cell line A549 is obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells are routinelycultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg,Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies,Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100μg/mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells are routinelypassaged by trysinization and dilution when they reach 90% confluence.Cells are seeded into 96-well plates (Falcon-Primaria #3872) at adensity of 7000 cells/well for treatment with the compound of theinvention.

Primary Mouse Hepatocytes

Primary mouse hepatocytes are prepared from CD-1 mice purchased fromCharles River Labs. Primary mouse hepatocytes are routinely cultured inHepatocyte Attachment Media (Invitrogen Life Technologies, Carlsbad,Calif.) supplemented with 10% Fetal Bovine Serum (Invitrogen LifeTechnologies, Carlsbad, Calif.), 250 nM dexamethasone (Sigma-AldrichCorporation, St. Louis, Mo.), 10 nM bovine insulin (Sigma-AldrichCorporation, St. Louis, Mo.). Cells are seeded into 96-well plates(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a densityof 4000-6000 cells/well for treatment with the oligomeric compounds ofthe invention.

EXAMPLE 22

Liposome-Mediated Treatment with Oligomeric Compounds of the Invention

When cells reach the desired confluency, they can be treated with theoligomeric compounds of the invention by liposome-mediated transfection.For cells grown in 96-well plates, wells are washed once with 200 μLOPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 100μL of OPTI-MEM™-1 containing 2.5 μg/mL LIPOFECTIN™ (Gibco BRL) and theoligomeric compounds of the invention at the desired finalconcentration. After 4 hours of treatment, the medium is replaced withfresh medium. Cells are harvested 16 hours after treatment with theoligomeric compounds of the invention for target mRNA expressionanalysis by real-time PCR.

EXAMPLE 23

Electroporation-Mediated Treatment with Oligomeric Compounds of theInvention

When the cells reach the desired confluency, they can be treated withthe oligomeric compounds of the invention by electorporation. Cells areelectroporated in the presence of the desired concentration of anoligomeric compound of the invention in 1 mm cuvettes at a density of1×10⁷ cells/mL, a voltage of 75V and a pulse length of 6 ms. Followingthe delivery of the electrical pulse, cells are replated for 16 to 24hours. Cells are then harvested for target mRNA expression analysis byreal-time PCR.

EXAMPLE 24

Apoptosis Assay

Caspase-3 activity is evaluated with an fluorometric HTS Caspase-3 assay(Oncogene Research Products, San Diego, Calif.) that detects cleavageafter aspartate residues in the peptide sequence (DEVD). The DEVDsubstrate is labeled with a fluorescent molecule, which exhibits a blueto green shift in fluorescence upon cleavage. Active caspase-3 intreated cells is measured by this assay according to the manufacturer'sinstructions. Following treatment with the oligomeric compounds of theinvention, 50 μL of assay buffer is added to each well, followed byaddition 20 μL of the caspase-3 fluorescent substrate conjugate. Dataare obtained in triplicate. Fluorescence in wells is immediatelydetected (excitation/emission 400/505 nm) using a fluorescent platereader (SpectraMAX GeminiXS, Molecular Devices, Sunnyvale, Calif.). Theplate is covered and incubated at 37° C. for an additional three hours,after which the fluorescence is again measured (excitation/emission400/505 nm). The value at time zero is subtracted from the measurementobtained at 3 hours. The measurement obtained from the untreated controlcells is designated as 100% activity.

EXAMPLE 25

Cell Proliferation and Viability Assay

Cell viability and proliferation are measured using the CyQuant CellProliferation Assay Kit (Molecular Probes, Eugene, Oreg.) utilizing theCyQuant GR green fluorescent dye which exhibits strong fluorescenceenhancement when bound to cellular nucleic acids. The assay is performedaccording to the manufacturer's instructions. After the treatment withone or more oligomeric compounds of the invention, the microplate isgently inverted to remove the medium from the wells, which are eachwashed once with 200 μL of phosphate-buffered saline. Plates are frozenat −70° C. and then thawed. A volume of 200 μL of the CyQUANT GRdye/cell-lysis buffer is added to each well. The microplate is incubatedfor 5 minutes at room temperature, protected from light. Data areobtained in triplicate. Fluorescence in wells is immediately detected(excitation/emission 480/520 nm) using a fluorescent plate reader(SpectraMAX GeminiXS, Molecular Devices, Sunnyvale, Calif.). Themeasurement obtained from the untreated control cells is designated as100% activity.

1. A composition comprising an antisense oligomeric compound and atleast one sense oligomeric compound wherein said sense oligomericcompound is shorter than said antisense oligomeric compound and is atleast partially complementary to and forms a duplex region with saidantisense oligomeric compound.
 2. The composition of claim 1 comprisingat least two sense oligomeric compounds that are each at least partiallycomplementary to said antisense oligomeric compound forming duplexregions between said at least two sense strands and said antisensestrand.
 3. The composition of claim 1 further comprising at least onecovalent linkage between said antisense oligomeric compound and said atleast one sense oligomeric compound.
 4. The composition of claim 3wherein one sense oligomeric compound is covalently linked to the5′-termini of said antisense oligomeric compound and another senseoligomeric compound is covalently linked to the 3′-termini of saidantisense oligomeric compound.
 5. The composition of claim 3 whereineach of said covalent linkages independently comprises a sequence ofnon-hybridizing linked nucleosides or a bifunctional linking group. 6.The composition of claim 5 wherein said bifunctional linking groupcomprises a polyethylene glycol linking group.
 7. The composition ofclaim 1 wherein said antisense oligomeric compound and each of said atleast one sense oligomeric compound comprises a plurality of linkednucleosides linked by internucleoside linking groups.
 8. The compositionof claim 7 wherein the conformational geometry of each of said linkednucleosides is 3′-endo.
 9. The composition of claim 8 wherein each ofsaid nucleosides having 3′-endo conformational geometry comprises a2′-substitutuent group.
 10. The composition of claim 9 wherein each2′-substituent group is, independently, —F, —OH, —O—CH₂CH₂—O—CH₃,—O—CH₃, —O—CH₂—CH═CH₂ or —O—CH₂—CH—CH₂—NH(R_(j)) where R_(j) is H orC₁-C₁₀ alkyl.
 11. The composition of claim 10 wherein each of said2′-substituent groups is, independently, —F, —OH, —O—CH₂CH₂—O—CH₃ or—O—CH₃.
 12. The composition of claim 7 wherein at least one of saidnucleosides has a 2′-OH group.
 13. The composition of claim 10 whereineach of said nucleosides are modified nucleosides having other than2′-OH groups.
 14. The composition of claim 8 wherein at least one ofsaid nucleosides is a bicyclic sugar nucleoside having a2′-O—(CH₂)_(n)-4′ bridge wherein n is 1 or
 2. 15. The composition ofclaim 8 wherein at least one of said nucleosides is a 4′-S sugarmodified nulcleoside.
 16. The composition of claim 7 wherein each ofsaid internucleoside linking groups is independently a phosphodiester ora phosphorothioate internucleoside linking group.
 17. The composition ofclaim 1 wherein said antisense oligomeric compound and each of said atleast one sense oligomeric compound optionally comprises a terminalphosphate group, a terminal stabilizing or capping group, a 3′-overhangor a conjugate group.
 18. The composition of claim 17 wherein the5′-terminus of said antisense oligomeric compound comprises a5′-phosphate group.
 19. The composition of claim 1 wherein saidantisense oligomeric compound has from about 15 to about 30 nucleobasesin length and each of said at least one sense oligomeric compound hasfrom about 2 to about 12 nucleobases in length.
 20. A method ofinhibiting gene expression comprising contacting one or more cells, atissue or an animal with a composition of claim 1.